Diagnostics and therapeutics for diseases associated with an IL-1 inflammatory haplotype

ABSTRACT

Methods and kits for determining whether a subject has or is predisposed to developing a disease which is associated with IL-1 polymorphisms and assays for identifying therapeutics for treating and/or preventing the development of these diseases are provided.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Ser. No. 09/845,129,filed Apr. 27, 2001, which is a continuation of U.S. Ser. No.09/345,217, filed Jun. 30, 1999 (now U.S. Pat. No. 6,268,142), whichclaims the benefit of Foreign Application No. PCT/GB98/01481, filed May21, 1998 and Foreign Application No. GB9711040.7, filed May 29, 1997;the entire contents of each are hereby incorporated by reference.

1. BACKGROUND OF THE INVENTION Genetics of the IL-1 Gene Cluster

[0002] The IL-1 gene cluster is on the long arm of chromosome 2 (2q13)and contains at least the genes for IL-1α (IL-1A), IL-1β (IL-1B), andthe IL-1 receptor antagonist (IL-1RN), within a region of 430 Kb(Nicklin, et al. (1994) Genomics, 19: 382-4). The agonist molecules,IL-1 α and IL-1β, have potent pro-inflammatory activity and are at thehead of many inflammatory cascades. Their actions, often via theinduction of other cytokines such as IL-6 and IL-8, lead to activationand recruitment of leukocytes into damaged tissue, local production ofvasoactive agents, fever response in the brain and hepatic acute phaseresponse. All three IL-1 molecules bind to type I and to type II IL-1receptors, but only the type I receptor transduces a signal to theinterior of the cell. In contrast, the type II receptor is shed from thecell membrane and acts as a decoy receptor. The receptor antagonist andthe type II receptor, therefore, are both anti-inflammatory in theiractions.

[0003] Inappropriate production of IL-1 plays a central role in thepathology of many autoimmune and inflammatory diseases, includingrheumatoid arthritis, inflammatory bowel disorder, psoriasis, and thelike. In addition, there are stable inter-individual differences in therates of production of IL-1, and some of this variation may be accountedfor by genetic differences at IL-1 gene loci. Thus, the IL-1 genes arereasonable candidates for determining part of the genetic susceptibilityto inflammatory diseases, most of which have a multifactorial etiologywith a polygenic component.

[0004] Certain alleles from the IL-1 gene cluster are known to beassociated with particular disease states. For example, IL-1RN (VNTR)allele 2 has been shown to be associated with osteoporosis (U.S. Pat.No. 5,698,399), nephropathy in diabetes mellitus (Blakemore, et al.(1996) Hum. Genet 97(3): 369-74), alopecia areata (Cork, et al., (1995)J. Invest. Dermatol. 104(5 Supp.): 15S-16S; Cork et al. (1996) DermatolClin 14: 671-8), Graves disease (Blakemore, et al. (1995) J. Clin.Endocrinol. 80(1): 111-5), systemic lupus erythematosus (Blakemore, etal. (1994) Arthritis Rheum. 37: 1380-85), lichen sclerosis (Clay, et al.(1994) Hum. Genet. 94: 407-10), and ulcerative colitis (Mansfield, etal. (1994) Gastoenterol. 106(3): 637-42)).

[0005] In addition, the IL-1A allele 2 from marker −889 and IL-1B (TaqI)allele 2 from marker +3954 have been found to be associated withperiodontal disease (U.S. Pat. No. 5,686,246; Kornman and diGiovine(1998) Ann Periodont 3: 327-38; Hart and Kornman (1997) Periodontol 200014: 202-15; Newman (1997) Compend Contin Educ Dent 18: 881-4; Kornman etal. (1997) J. Clin Periodontol 24: 72-77). The IL-1A allele 2 frommarker −889 has also been found to be associated with juvenile chronicarthritis, particularly chronic iridocyclitis (McDowell, et al. (1995)Arthritis Rheum. 38: 221-28). The IL-1B (TaqI) allele 2 from marker+3954 of IL-1B has also been found to be associated with psoriasis andinsulin dependent diabetes in DR3/4 patients (di Giovine, et al. (1995)Cytokine 7: 606; Pociot, et al. (1 992) Eur J. Clin. Invest. 22:396-402). Additionally, the IL-1RN (VNTR) allele 1 has been found to beassociated with diabetic retinopathy (see U.S. Ser. No. 09/037472, andPCT/GB97/02790). Furthermore allele 2 of IL-1RN (VNTR) has been found tobe associated with ulcerative colitis in Caucasian populations fromNorth America and Europe (Mansfield, J. et al., (1994) Gastroenterology106: 637-42). Interestingly, this association is particularly strongwithin populations of ethnically related Ashkenazi Jews (PCTWO97/25445).

Genotype Screening

[0006] Traditional methods for the screening of heritable diseases havedepended on either the identification of abnormal gene products (e.g.,sickle cell anemia) or an abnormal phenotype (e.g., mental retardation).These methods are of limited utility for heritable diseases with lateonset and no easily identifiable phenotypes such as, for example,vascular disease. With the development of simple and inexpensive geneticscreening methodology, it is now possible to identify polymorphisms thatindicate a propensity to develop disease, even when the disease is ofpolygenic origin. The number of diseases that can be screened bymolecular biological methods continues to grow with increasedunderstanding of the genetic basis of multifactorial disorders.

[0007] Genetic screening (also called genotyping or molecularscreening), can be broadly defined as testing to determine if a patienthas mutations (alleles or polymorphisms) that either cause a diseasestate or are “linked” to the mutation causing a disease state. Linkagerefers to the phenomenon th DNA sequences which are close together inthe genome have a tendency to be inherited together. Two sequences maybe linked because of some selective advantage of co-inheritance. Moretypically, however, two polymorphic sequences are co-inherited becauseof the relative infrequency with which meiotic recombination eventsoccur within the region between the two polymorphisms. The co-inheritedpolymorphic alleles are said to be in linkage disequilibrium with oneanother because, in a given human population, they tend to either bothoccur together or else not occur at all in any particular member of thepopulation. Indeed, where multiple polymorphisms in a given chromosomalregion are found to be in linkage disequilibrium with one another, theydefine a quasi-stable genetic “haplotype.” In contrast, recombinationevents occurring between two polymorphic loci cause them to becomeseparated onto distinct homologous chromosomes. If meiotic recombinationbetween two physically linked polymorphisms occurs frequently enough,the two polymorphisms will appear to segregate independently and aresaid to be in linkage equilibrium.

[0008] While the frequency of meiotic recombination between two markersis generally proportional to the physical distance between them on thechromosome, the occurrence of “hot spots” as well as regions ofrepressed chromosomal recombination can result in discrepancies betweenthe physical and recombinational distance between two markers. Thus, incertain chromosomal regions, multiple polymorphic loci spanning a broadchromosomal domain may be in linkage disequilibrium with one another,and thereby define a broad-spanning genetic haplotype. Furthermore,where a disease-causing mutation is found within or in linkage with thishaplotype, one or more polymorphic alleles of the haplotype can be usedas a diagnostic or prognostic indicator of the likelihood of developingthe disease. This association between otherwise benign polymorphisms anda disease-causing polymorphism occurs if the disease mutation arose inthe recent past, so that sufficient time has not elapsed for equilibriumto be achieved through recombination events. Therefore identification ofa human haplotype which spans or is linked to a disease-causingmutational change, serves as a predictive measure of an individual'slikelihood of having inherited that disease-causing mutation.Importantly, such prognostic or diagnostic procedures can be utilizedwithout necessitating the identification and isolation of the actualdisease-causing lesion. This is significant because the precisedetermination of the molecular defect involved in a disease process canbe difficult and laborious, especially in the case of multifactorialdiseases such as inflammatory disorders.

[0009] Indeed, the statistical correlation between an inflammatorydisorder and an IL-1 polymorphism does not necessarily indicate that thepolymorphism directly causes the disorder. Rather the correlatedpolymorphism may be a benign allelic variant which is linked to (i.e. inlinkage disequilibrium with) a disorder-causing mutation which hasoccurred in the recent human evolutionary past, so that sufficient timehas not elapsed for equilibrium to be achieved through recombinationevents in the intervening chromosomal segment. Thus, for the purposes ofdiagnostic and prognostic assays for a particular disease, detection ofa polymorphic allele associated with that disease can be utilizedwithout consideration of whether the polymorphism is directly involvedin the etiology of the disease. Furthermore, where a given benignpolymorphic locus is in linkage disequilibrium with an apparentdisease-causing polymorphic locus, still other polymorphic loci whichare in linkage disequilibrium with the benign polymorphic locus are alsolikely to be in linkage disequilibrium with the disease-causingpolymorphic locus. Thus these other polymorphic loci will also beprognostic or diagnostic of the likelihood of having inherited thedisease-causing polymorphic locus. Indeed, a broad-spanning humanhaplotype (describing the typical pattern of co-inheritance of allelesof a set of linked polymorphic markers) can be targeted for diagnosticpurposes once an association has been drawn between a particular diseaseor condition and a corresponding human haplotype. Thus, thedetermination of an individual's likelihood for developing a particulardisease of condition can be made by characterizing one or moredisease-associated polymorphic alleles (or even one or moredisease-associated haplotypes) without necessarily determining orcharacterizing the causative genetic variation.

2. SUMMARY OF THE INVENTION

[0010] In one aspect, the present invention provides novel methods andkits for determining whether a subject has or is predisposed todeveloping a disease or condition that is associated with an IL-1polymorphism. In one embodiment, the method comprises determiningwhether the subject's nucleic acids contain a marker or allelecomprising an IL-1 inflammatory haplotype. In a preferred embodiment,the IL-1 inflammatory haplotype is indicative of increased Il-1 agonist(e.g. IL-1 (44112332)). In another preferred embodiment, the IL-1inflammatory haplotype is indicative of decreased IL-1 receptorantagonist activity (e.g. IL-1 (33441461)).

[0011] An allele comprising an IL-1 inflammatory haplotype can bedetected by any of a variety of available techniques, including: 1)performing a hybridization reaction between a nucleic acid sample and aprobe that is capable of hybridizing to the allele; 2) sequencing atleast a portion of the allele; or 3) determining the electrophoreticmobility of the allele or fragments thereof (e.g., fragments generatedby endonuclease digestion). The allele can optionally be subjected to anamplification step prior to performance of the detection step. Preferredamplification methods are selected from the group consisting of: thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), stranddisplacement amplification (SDA), cloning, and variations of the above(e.g. RT-PCR and allele specific amplification). Oligonucleotidesnecessary for amplification may be selected, for example, from withinthe IL-1 gene loci, either flanking the marker of interest (as requiredfor PCR amplification) or directly overlapping the marker (as in ASOhybridization). In a particularly preferred embodiment, the sample ishybridized with a set of primers, which hybridize 5′ and 3′ in a senseor antisense sequence to the vascular disease associated allele, and issubjected to a PCR amplification.

[0012] An allele comprising an IL-1 inflammatory haplotype may also bedetected indirectly, e.g. by analyzing the protein product encoded bythe DNA. For example, where the marker in question results in thetranslation of a mutant protein, the protein can be detected by any of avariety of protein detection methods. Such methods includeimmunodetection and biochemical tests, such as size fractionation, wherethe protein has a change in apparent molecular weight either throughtruncation, elongation, altered folding or altered post-translationalmodifications.

[0013] In another aspect, the invention features kits for performing theabove-described assays. The kit can include a nucleic acid samplecollection means and a means for determining whether a subject carriesat least one allele comprising an IL-1 inflammatory haplotype. The kitmay also contain a control sample either positive or negative or astandard and/or an algorithmic device for assessing the results andadditional reagents and components including: DNA amplificationreagents, DNA polymerase, nucleic acid amplification reagents,restrictive enzymes, buffers, a nucleic acid sampling device, DNApurification device, deoxynucleotides, oligonucleotides (e.g. probes andprimers) etc.

[0014] As described above, the control may be a positive or negativecontrol. Further, the control sample may contain the positive (ornegative) products of the allele detection technique employed. Forexample, where the allele detection technique is PCR amplification,followed by size fractionation, the control sample may comprise DNAfragments of the appropriate size. Likewise, where the allele detectiontechnique involves detection of a mutated protein, the control samplemay comprise a sample of mutated protein. However, it is preferred thatthe control sample comprises the material to be tested. For example, thecontrols may be a sample of genomic DNA or a cloned portion of the IL-1gene cluster. Preferably, however, the control sample is a highlypurified sample of genomic DNA where the sample to be tested is genomicDNA.

[0015] The oligonucleotides present in said kit may be used foramplification of the region of interest or for direct allele specificoligonucleotide (ASO) hybridization to the markers in question. Thus,the oligonucleotides may either flank the marker of interest (asrequired for PCR amplification) or directly overlap the marker (as inASO hybridization).

[0016] Information obtained using the assays and kits described herein(alone or in conjunction with information on another genetic defect orenvironmental factor, which contributes to the disease or condition thatis associated with an IL-1 inflammatory haplotype) is useful fordetermining whether a non-symptomatic subject has or is likely todevelop the particular disease or condition. In addition, theinformation can allow a more customized approach to preventing the onsetor progression of the disease or condition. For example, thisinformation can enable a clinician to more effectively prescribe atherapy that will address the molecular basis of the disease orcondition.

[0017] In yet a further aspect, the invention features methods fortreating or preventing the development of a disease or condition that isassociated with an IL-1 inflammatory haplotype in a subject byadministering to the subject an appropriate therapeutic of theinvention. In still another aspect, the invention provides in vitro orin vivo assays for screening test compounds to identify therapeutics fortreating or preventing the development of a disease or condition that isassociated with an IL-1 inflammatory haplotype. In one embodiment, theassay comprises contacting a cell transfected with a causative mutationthat is operably linked to an appropriate promoter with a test compoundand determining the level of expression of a protein in the cell in thepresence and in the absence of the test compound. In a preferredembodiment, the causative mutation results in decreased production ofIL-1 receptor antagonist, and increased production of the IL-1 receptorantagonist in the presence of the test compound indicates that thecompound is an agonist of IL-1 receptor antagonist activity. In anotherpreferred embodiment, the causative mutation results in increasedproduction of IL-1α or IL-1β, and decreased production of IL-1α or IL-1βin the presence of the test compound indicates that the compound is anantagonist of IL-1α or IL-1β activity. In another embodiment, theinvention features transgenic non-human animals and their use inidentifying antagonists of IL-1α or IL-1β activity or agonists of IL-1Raactivity.

[0018] Other embodiments and advantages of the invention are set forthin the following detailed description and claims.

3. BRIEF DESCRIPTION OF THE FIGURES

[0019]FIG. 1 is a schematic depiction of the IL-1 gene cluster includinga few polymorphic markers.

[0020]FIG. 2 is a graph which plots the correlation betweendisequilibrium values and physical distance as described herein.

[0021]FIG. 3 shows the nucleic acid sequence for IL-1A (GEN X03833; SEQID No. 1).

[0022]FIG. 4 shows the nucleic acid sequence for IL-1B (GEN X04500; SEQID No. 2).

[0023]FIG. 5 shows the nucleic acid sequence for the secreted IL-1RN(GEN X64532; SEQ ID No. 3).

4. DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions

[0024] For convenience, the meaning of certain terms and phrasesemployed in the specification, examples, and appended claims is providedbelow.

[0025] The term “allele” refers to the different sequence variants foundat different polymorphic regions. For example, IL-1RN (VNTR) has atleast five different alleles. The sequence variants may be single ormultiple base changes, including without limitation insertions,deletions, or substitutions, or may be a variable number of sequencerepeats.

[0026] The term “allelic pattern” refers to the identity of an allele oralleles at one or more polymorphic regions. For example, an allelicpattern may consist of a single allele at a polymorphic site, as forIL-1RN (VNTR) allele 1, which is an allelic pattern having at least onecopy of IL-1RN allele I at the VNTR of the IL-1RN gene loci.Alternatively, an allelic pattern may consist of either a homozygous orheterozygous state at a single polymorphic site. For example, IL1-RN(VNTR) allele 2,2 is an allelic pattern in which there are two copies ofthe second allele at the VNTR marker of IL-1RN that corresponds to thehomozygous IL-RN (VNTR) allele 2 state. Alternatively, an allelicpattern may consist of the identity of alleles at more than onepolymorphic site.

[0027] The term “antibody ” as used herein is intended to refer to abinding agent including a whole antibody or a binding fragment thereofwhich is specifically reactive with an IL-1 polypeptide. Antibodies canbe fragmented using conventional techniques and the fragments screenedfor utility in the same manner as described above for whole antibodies.For example, F(ab)2 fragments can be generated by treating an antibodywith pepsin. The resulting F(ab)2 fragment can be treated to reducedisulfide bridges to produce Fab fragments. The antibody of the presentinvention is further intended to include bispecific, single-chain, andchimeric and humanized molecules having affinity for an IL-1Bpolypeptide conferred by at least one CDR region of the antibody.

[0028] “Biological activity” or “bioactivity” or “activity” or“biological function”, which are used interchangeably, for the purposesherein means an effector or antigenic function that is directly orindirectly performed by an IL-1 polypeptide (whether in its native ordenatured conformation), or by any subsequence thereof. Biologicalactivities include binding to a target peptide, e.g., an IL-1 receptor.An IL-1 bioactivity can be modulated by directly affecting an IL-1polypeptide. Alternatively, an IL-1 bioactivity can be modulated bymodulating the level of an IL-1 polypeptide, such as by modulatingexpression of an IL-1 gene.

[0029] As used herein the term “bioactive fragment of an IL-1polypeptide” refers to a fragment of a full-length IL-1 polypeptide,wherein the fragment specifically mimics or antagonizes the activity ofa wild-type IL-1 polypeptide. The bioactive fragment preferably is afragment capable of interacting with an interleukin receptor.

[0030] The term “an aberrant activity”, as applied to an activity of apolypeptide such as IL-1, refers to an activity which differs from theactivity of the wild-type or native polypeptide or which differs fromthe activity of the polypeptide in a healthy subject. An activity of apolypeptide can be aberrant because it is stronger than the activity ofits native counterpart. Alternatively, an activity can be aberrantbecause it is weaker or absent relative to the activity of its nativecounterpart. An aberrant activity can also be a change in an activity.For example an aberrant polypeptide can interact with a different targetpeptide. A cell can have an aberrant IL-1 activity due to overexpressionor underexpression of an IL-1 locus gene encoding an IL-1 locuspolypeptide.

[0031] “Cells”, “host cells” or “recombinant host cells” are terms usedinterchangeably herein to refer not only to the particular subject cell,but to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact be identicalto the parent cell, but are still included within the scope of the termas used herein.

[0032] A “chimera,” “mosaic,” “chimeric mammal” and the like, refers toa transgenic mammal with a knock-out or knock-in construct in at leastsome of its genome-containing cells.

[0033] The terms “control” or “control sample” refer to any sampleappropriate to the detection technique employed. The control sample maycontain the products of the allele detection technique employed or thematerial to be tested. Further, the controls may be positive or negativecontrols. By way of example, where the allele detection technique is PCRamplification, followed by size fractionation, the control sample maycomprise DNA fragments of an appropriate size. Likewise, where theallele detection technique involves detection of a mutated protein, thecontrol sample may comprise a sample of a mutant protein. However, it ispreferred that the control sample comprises the material to be tested.For example, the controls may be a sample of genomic DNA or a clonedportion of the IL-1 gene cluster. However, where the sample to be testedis genomic DNA, the control sample is preferably a highly purifiedsample of genomic DNA.

[0034] The phrase “diseases and conditions associated with IL-1polymorphisms” refers to a variety of diseases or conditions, thesusceptibility to which can be indicated in a subject based on theidentification of one or more alleles within the IL-1 complex. Examplesinclude: inflammatory or degenerative disease, including: SystemicInflammatory Response (SIRS); Alzheimer's Disease (and associatedconditions and symptoms including: chronic neuroinflammation, glialactivation; increased microglia; neuritic plaque formation; and responseto therapy); Amylotropic Lateral Sclerosis (ALS), arthritis (andassociated conditions and symptoms including: acute joint inflammation,antigen-induced arthritis, arthritis associated with chronic lymphocyticthyroiditis, collagen-induced arthitis, juvenile chronic arthritis;juvenile rheumatoid arthritis, osteoarthritis, prognosis andstreptococcus-induced arthritis), asthma (and associated conditions andsymptoms, including: bronchial asthma; chronic obstructive airwaydisease; chronic obstructive pulmonary disease, juvenile asthma andoccupational asthma); cardiovascular diseases (and associated conditionsand symptoms, including atherosclerosis; autoimmune myocarditis, chroniccardiac hypoxia, congestive heart failure, coronary artery disease,cardiomyopathy and cardiac cell dysfunction, including: aortic smoothmuscle cell activation; cardiac cell apoptosis; and immunomodulation ofcardiac cell function; diabetes and associated conditions and symptoms,including autoimmune diabetes, insulin-dependent (Type 1) diabetes,diabetic periodontitis, diabetic retinopathy, and diabetic nephropathy);gastrointestinal inflammations (and related conditions and symptoms,including celiac disease, associated osteopenia, chronic colitis,Crohn's disease, inflammatory bowel disease and ulcerative colitis);gastric ulcers; hepatic inflammations, cholesterol gallstones andhepatic fibrosis, HIV infection (and associated conditions and symptoms,including degenerative responses, neurodegenerative responses, and HIVassociated Hodgkin's Disease), Kawasaki's Syndrome (and associateddiseases and conditions, including mucocutaneous lymph node syndrome,cervical lymphadenopathy, coronary artery lesions, edema, fever,increased leukocytes, mild anemia, skin peeling, rash, conjunctivaredness, thrombocytosis; multiple sclerosis, nephropathies (andassociated diseases and conditions, including diabetic nephropathy,endstage renal disease, glomerulonephritis, Goodpasture's syndrome,hemodialysis survival and renal ischemic reperfusion injury),neurodegenerative diseases (and associated diseases and conditions,including acute neurodegeneration, induction of IL-1 in aging andneurodegenerative disease, IL-1 induced plasticity of hypothalamicneurons and chronic stress hyperresponsiveness), Qphthalmopathies (andassociated diseases and conditions, including diabetic retinopathy,Graves' Ophthalmopathy, and uveitis, osteoporosis (and associateddiseases and conditions, including alveolar, femoral, radial, vertebralor wrist bone loss or fracture incidence, postmenopausal bone loss,mass, fracture incidence or rate of bone loss), otitis media (adult orpediatric), pancreatis or pancreatic acinitis, periodontal disease (andassociated diseases and conditions, including adult, early onset anddiabetic); pulmonary diseases, including chronic lung disease, chronicsinusitis, hyaline membrane disease, hypoxia and pulmonary disease inSIDS; restenosis; rheumatism including rheumatoid arthritis, rheumaticaschoff bodies, rheumatic diseases and rheumatic myocarditis;thyroiditis including chronic lymphocytic thyroiditis; urinary tractinfections including chronic prostatitis, chronic pelvic pain syndromeand urolithiasis. Immunological disorders, including autoimmunediseases, such as alopecia aerata, autoimmune myocarditis, Graves'disease, Graves ophthalmopathy, lichen sclerosis, multiple sclerosis,psoriasis, systemic lupus erythematosus, systemic sclerosis, thyroiddiseases (e.g. goiter and struma lymphomatosa (Hashimoto's thyroiditis,lymphadenoid goiter), sleep disorders and chronic fatigue syndrome andobesity (non-diabetic or associated with diabetes). Resistance toinfectious diseases, such as Leishmaniasis, Leprosy, Lyme Disease, LymeCarditis, malaria, cerebral malaria, meningititis, tubulointestitialnephritis associated with malaria), which are caused by bacteria,viruses (e.g. cytomegalovirus, encephalitis, Epstein-Barr Virus, HumanImmunodeficiency Virus, Influenza Virus) or protozoans (e.g., Plasmodiumfalciparum, trypanosomes). Response to trauma, including cerebral trauma(including strokes and ischemias, encephalitis, encephalopathies,epilepsy, perinatal brain injury, prolonged febrile seizures, SIDS andsubarachnoid hemorrhage), low birth weight (e.g. cerebral palsy), lunginjury (acute hemorrhagic lung injury, Goodpasture's syndrome, acuteischemic reperfusion), myocardial dysfunction, caused by occupationaland environmental pollutants (e.g. susceptibility to toxic oil syndromesilicosis), radiation trauma, and efficiency of wound healing responses(e.g. burn or thermal wounds, chronic wounds, surgical wounds and spinalcord injuries). Susceptibility to neoplasias, including breast cancerassociated osteolytic metastasis, cachexia, colorectal cancer,hyperproliferative diseases, Hodgkin's disease, leukemias, lymphomas,metabolic diseases and tumors, metastases, myeolomas, and variouscancers (including breast prostate ovarian, colon, lung, etc), anorexiaand cachexia. Hormonal regulation including fertility/fecundity,likelihood of a pregnancy, incidence of preterm labor, prenatal andneonatal complications including preterm low birth weight, cerebralpalsy, septicemia, hypothyroxinernia, oxygen dependence, cranialabnormality, early onset menopause. A subject's response to transplant(rejection or acceptance), acute phase response (e.g. febrile response),general inflammatory response, acute respiratory distress response,acute systemic inflammatory response, wound healing, adhesion,immunoinflammatory response, neuroendocrine response, fever developmentand resistance, acute-phase response, stress response, diseasesusceptibility, repetitive motion stress, tennis elbow, and painmanagement and response.

[0035] The phrases “disruption of the gene” and “targeted disruption” orany similar phrase refers to the site specific interruption of a nativeDNA sequence so as to prevent expression of that gene in the cell ascompared to the wild-type copy of the gene. The interruption may becaused by deletions, insertions or modifications to the gene, or anycombination thereof.

[0036] The term “haplotype” as used herein is intended to refer to a setof alleles that are inherited together as a group (are in linkagedisequilibrium) at statistically significant levels (p_(corr)<0.05). Asused herein, the phrase “an IL-1 haplotype” refers to a haplotype in theIL-1 loci. An IL-1 inflammatory or proinflammatory haplotype refers to ahaplotype that is indicative of increased agonist and/or decreasedantagonist activities.

[0037] The terms “IL-1 gene cluster” and “IL-1 loci” as used hereininclude all the nucleic acid at or near the 2q13 region of chromosome 2,including at least the IL-1A, IL-1B and IL-1RN genes and any otherlinked sequences. (Nicklin et al., Genomics 19: 382-84, 1994). The terms“IL-1A”, “IL-1B”, and “IL-1RN” as used herein refer to the genes codingfor IL-1, IL-1, and IL-1 receptor antagonist, respectively. The geneaccession number for IL-1A, IL-1B, and IL-1RN are X03833, X04500, andX64532, respectively.

[0038] “IL-1 functional mutation” refers to a mutation within the IL-1gene cluster that results in an altered phenotype (i.e. effects thefunction of an IL-1 gene or protein). Examples include: IL-1A(+4845)allele 2, IL-1B (+3954) allele 2, IL-1B (+6912) allele 2 and IL-1RN(+2018) allele 2.

[0039] “IL-1X (Z) allele Y” refers to a particular allelic form,designated Y, occurring at an IL-1 locus polymorphic site in gene X,wherein X is IL-1A, B, or RN and positioned at or near nucleotide Z,wherein nucleotide Z is numbered relative to the major transcriptionalstart site, which is nucleotide +1, of the particular IL-1 gene X. Asfurther used herein, the term “IL-1X allele (Z)” refers to all allelesof an IL-1 polymorphic site in gene X positioned at or near nucleotideZ. For example, the term “IL-1RN (+2018) allele” refers to alternativeforms of the IL-1RN gene at marker +2018. “IL-1RN (+2018) allele 1”refers to a form of the IL-1RN gene which contains a cytosine (C) atposition +2018 of the sense strand. Clay et al., Hum. Genet. 97:723-26,1996. “IL-1RN (+2018) allele 2” refers to a form of the IL-1RN genewhich contains a thymine (T) at position +2018 of the plus strand. Whena subject has two identical IL-1RN alleles, the subject is said to behomozygous, or to have the homozygous state. When a subject has twodifferent IL-1RN alleles, the subject is said to be heterozygous, or tohave the heterozygous state. The term “IL-1RN (+2018) allele 2,2” refersto the homozygous IL-1RN (+2018) allele 2 state. Conversely, the term“IL-1RN (+2018) allele 1,1” refers to the homozygous IL-1RN (+2018)allele 1 state. The term “IL-1RN (+2018) allele 1,2” refers to theheterozygous allele 1 and 2 state.

[0040] “IL-1 related” as used herein is meant to include all genesrelated to the human IL-1 locus genes on human chromosome 2 (2q 12-14).These include IL-1 genes of the human IL-1 gene cluster located atchromosome 2 (2q 13-14) which include: the IL-1A gene which encodesinterleukin-1α, the IL-1B gene which encodes interleukin-1β, and theIL-1RN (or IL-1ra) gene which encodes the interleukin-1 receptorantagonist. Furthermore these IL-1 related genes include the type I andtype II human IL-1 receptor genes located on human chromosome 2 (2q12)and their mouse homologs located on mouse chromosome 1 at position 19.5cM. Interleukin-1α, interleukin-1β, and interleukin-1RN are related inso much as they all bind to IL-1 type I receptors, however onlyinterleukin-1α and interleukin-1β are agonist ligands which activateIL-1 type I receptors, while interleukin-1RN is a naturally occurringantagonist ligand. Where the term “IL-1” is used in reference to a geneproduct or polypeptide, it is meant to refer to all gene productsencoded by the interleukin-1 locus on human chromosome 2 (2q 12-14) andtheir corresponding homologs from other species or functional variantsthereof. The term IL-1 thus includes secreted polypeptides which promotean inflammatory response, such as IL-1α and IL-1β, as well as a secretedpolypeptide which antagonize inflammatory responses, such as IL-1receptor antagonist and the IL-1 type II (decoy) receptor.

[0041] An “IL-1 receptor” or “IL-1R” refers to various cell membranebound protein receptors capable of binding to and/or transducing asignal from an IL-1 locus-encoded ligand. The term applies to any of theproteins which are capable of binding interleukin-1 (IL-1) moleculesand, in their native configuration as mammalian plasma membraneproteins, presumably play a role in transducing the signal provided byIL-1 to a cell. As used herein, the term includes analogs of nativeproteins with IL-1-binding or signal transducing activity. Examplesinclude the human and murine IL-1 receptors described in U.S. Pat. No.4,968,607. The term “IL-1 nucleic acid” refers to a nucleic acidencoding an IL-1 protein.

[0042] An “IL-1 polypeptide” and “IL-1 protein” are intended toencompass polypeptides comprising the amino acid sequence encoded by theIL-1 genomic DNA sequences shown in FIGS. 1, 2, and 3, or fragmentsthereof, and homologs thereof and include agonist and antagonistpolypeptides.

[0043] “Increased risk” refers to a statistically higher frequency ofoccurrence of the disease or condition in an individual carrying aparticular polymorphic allele in comparison to the frequency ofoccurrence of the disease or condition in a member of a population thatdoes not carry the particular polymorphic allele.

[0044] The term “interact” as used herein is meant to include detectablerelationships or associations (e.g. biochemical interactions) betweenmolecules, such as interactions between protein-protein, protein-nucleicacid, nucleic acid-nucleic acid and protein-small molecule or nucleicacid-small molecule in nature.

[0045] The term “isolated” as used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs, orRNAs, respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject IL-1 polypeptides preferably includes no more than 10 kilobases(kb) of nucleic acid sequence which naturally immediately flanks theIL-1 gene in genomic DNA, more preferably no more than 5 kb of suchnaturally occurring flanking sequences, and most preferably less than1.5 kb of such naturally occurring flanking sequence. The term isolatedas used herein also refers to a nucleic acid or peptide that issubstantially free of cellular material, viral material, or culturemedium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized. Moreover, an“isolated nucleic acid” is meant to include nucleic acid fragments whichare not naturally occurring as fragments and would not be found in thenatural state. The term “isolated” is also used herein to refer topolypeptides which are isolated from other cellular proteins and ismeant to encompass both purified and recombinant polypeptides.

[0046] A “knock-in” transgenic animal refers to an animal that has had amodified gene introduced into its genome and the modified gene can be ofexogenous or endogenous origin.

[0047] A “knock-out” transgenic animal refers to an animal in whichthere is partial or complete suppression of the expression of anendogenous gene (e.g, based on deletion of at least a portion of thegene, replacement of at least a portion of the gene with a secondsequence, introduction of stop codons, the mutation of bases encodingcritical amino acids, or the removal of an intron junction, etc.).

[0048] A “knock-out construct” refers to a nucleic acid sequence thatcan be used to decrease or suppress expression of a protein encoded byendogenous DNA sequences in a cell. In a simple example, the knock-outconstruct is comprised of a gene, such as the IL-1RN gene, with adeletion in a critical portion of the gene, so that active proteincannot be expressed therefrom. Alternatively, a number of terminationcodons can be added to the native gene to cause early termination of theprotein or an intron junction can be inactivated. In a typical knock-outconstruct, some portion of the gene is replaced with a selectable marker(such as the neo gene) so that the gene can be represented as follows:IL-1RN 5′/neo/IL-1RN 3′, where IL-1RN5′ and IL-1RN 3′, refer to genomicor cDNA sequences which are, respectively, upstream and downstreamrelative to a portion of the IL-1RN gene and where neo refers to aneomycin resistance gene. In another knock-out construct, a secondselectable marker is added in a flanking position so that the gene canbe represented as: IL-1RN /neo/IL-1RN/TK, where TK is a thymidine kinasegene which can be added to either the IL-1RN 5′ or the IL-1RN 3′sequence of the preceding construct and which further can be selectedagainst (i.e. is a negative selectable marker) in appropriate media.This two-marker construct allows the selection of homologousrecombination events, which removes the flanking TK marker, fromnon-homologous recombination events which typically retain the TKsequences. The gene deletion and/or replacement can be from the exons,introns, especially intron junctions, and/or the regulatory regions suchas promoters.

[0049] “Linkage disequilibrium” refers to co-inheritance of two allelesat frequencies greater than would be expected from the separatefrequencies of occurrence of each allele in a given control population.The expected frequency of occurrence of two alleles that are inheritedindependently is the frequency of the first allele multiplied by thefrequency of the second allele. Alleles that co-occur at expectedfrequencies are said to be in “linkage disequilibrium”. The cause oflinkage disequilibrium is often unclear. It can be due to selection forcertain allele combinations or to recent admixture of geneticallyheterogeneous populations. In addition, in the case of markers that arevery tightly linked to a disease gene, an association of an allele (orgroup of linked alleles) with the disease gene is expected if thedisease mutation occurred in the recent past, so that sufficient timehas not elapsed for equilibrium to be achieved through recombinationevents in the specific chromosomal region. When referring to allelicpatterns that are comprised of more than one allele, a first allelicpattern is in linkage disequilibrium with a second allelic pattern ifall the alleles that comprise the first allelic pattern are in linkagedisequilibrium with at least one of the alleles of the second allelicpattern. An example of linkage disequilibrium is that which occursbetween the alleles at the IL-1RN (+2018) and IL-1RN (VNTR) polymorphicsites. The two alleles at IL-1RN (+2018) are 100% in linkagedisequilibrium with the two most frequent alleles of IL-1RN (VNTR),which are allele 1 and allele 2.

[0050] The term “marker” refers to a sequence in the genome that isknown to vary among individuals. For example, the IL-1RN gene has amarker that consists of a variable number of tandem repeats (VNTR).

[0051] A “mutated gene” or “mutation” or “functional mutation” refers toan allelic form of a gene, which is capable of altering the phenotype ofa subject having the mutated gene relative to a subject which does nothave the mutated gene. The altered phenotype caused by a mutation can becorrected or compensated for by certain agents. If a subject must behomozygous for this mutation to have an altered phenotype, the mutationis said to be recessive. If one copy of the mutated gene is sufficientto alter the phenotype of the subject, the mutation is said to bedominant. If a subject has one copy of the mutated gene and has aphenotype that is intermediate between that of a homozygous and that ofa heterozygous subject (for that gene), the mutation is said to beco-dominant.

[0052] A “non-human animal” of the invention includes mammals such asrodents, non-human primates, sheep, dogs, cows, goats, etc. amphibians,such a s members of the Xenopus genus, and transgenic avians (e.g.chickens, birds, etc.). The term “chimeric animal” is used herein torefer to animals in which the recombinant gene is found, or in which therecombinant gene is expressed in some but not all cells of the animal.The term “tissue-specific chimeric animal” indicates that one of therecombinant IL-1 genes is present and/or expressed or disrupted in sometissues but not others. The term “non-human mammal” refers to any memberof the class Mammalia, except for humans.

[0053] As used herein, the term “nucleic acid” refers to polynucleotidesor oligonucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA). The term should also be understoodto include, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs (e.g. peptide nucleic acids) and as applicable to theembodiment being described, single (sense or antisense) anddouble-stranded polynucleotides.

[0054] The term “polymorphism” refers to the coexistence of more thanone form of a gene or portion (e.g., allelic variant) thereof. A portionof a gene of which there are at least two different forms, i.e., twodifferent nucleotide sequences, is referred to as a “polymorphic regionof a gene”. A specific genetic sequence at a polymorphic region of agene is an allele. A polymorphic region can be a single nucleotide, theidentity of which differs in different alleles. A polymorphic region canalso be several nucleotides long.

[0055] The term “propensity to disease,” also “predisposition” or“susceptibility” to disease or any similar phrase, means that certainalleles are hereby discovered to be associated with or predictive of asubject's incidence of developing a particular disease (e.g. a vasculardisease). The alleles are thus over-represented in frequency inindividuals with disease as compared to healthy individuals. Thus, thesealleles can be used to predict disease even in pre-symptomatic orpre-diseased individuals.

[0056] “Small molecule” as used herein, is meant to refer to acomposition, which has a molecular weight of less than about 5 kD andmost preferably less than about 4 kD. Small molecules can be nucleicacids, peptides, peptidomimetics, carbohydrates, lipids or other organicor inorganic molecules.

[0057] As used herein, the term “specifically hybridizes” or“specifically detects” refers to the ability of a nucleic acid moleculeto hybridize to at least approximately 6 consecutive nucleotides of asample nucleic acid.

[0058] “Transcriptional regulatory sequence” is a generic term usedthroughout the specification to refer to DNA sequences, such asinitiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they are operablylinked.

[0059] As used herein, the term “transgene” means a nucleic acidsequence (encoding, e.g., one of the IL-1 polypeptides, or an antisensetranscript thereto) which has been introduced into a cell. A transgenecould be partly or entirely heterologous, i.e., foreign, to thetransgenic animal or cell into which it is introduced, or, is homologousto an endogenous gene of the transgenic animal or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe animal's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can also be present in a cell in the form of anepisome. A transgene can include one or more transcriptional regulatorysequences and any other nucleic acid, such as introns, that may benecessary for optimal expression of a selected nucleic acid.

[0060] A “transgenic animal” refers to any animal, preferably anon-human mammal, bird or an amphibian, in which one or more of thecells of the animal contain heterologous nucleic acid introduced by wayof human intervention, such as by transgenic techniques well known inthe art. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.This molecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of an IL-1 polypeptide, e.g. either agonistic orantagonistic forms. However, transgenic animals in which the recombinantgene is silent are also contemplated, as for example, the FLP or CRErecombinase dependent constructs described below. Moreover, “transgenicanimal” also includes those recombinant animals in which gene disruptionof one or more genes is caused by human intervention, including bothrecombination and antisense techniques. The term is intended to includeall progeny generations. Thus, the founder animal and all F1, F2, F3,and so on, progeny thereof are included.

[0061] The term “treating” as used herein is intended to encompasscuring as well as ameliorating at least one symptom of a condition ordisease.

[0062] The term “vector” refers to a nucleic acid molecule, which iscapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/or expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

[0063] The term “wild-type allele” refers to an allele of a gene which,when present in two copies in a subject results in a wild-typephenotype. There can be several different wild-type alleles of aspecific gene, since certain nucleotide changes in a gene may not affectthe phenotype of a subject having two copies of the gene with thenucleotide changes.

4.2 Predictive Medicine 4.2.1. IL-1 Inflammatory Haplotypes and TheirAssociation with Certain Diseases or Conditions

[0064] The present invention is based at least in part, on theidentification of certain inflammatory haplotype patterns and theassociation (to a statistically significant extent) of these patternswith the development of certain diseases or conditions. Therefore,detection of the alleles comprising a haplotype, alone or in conjunctionwith another means in a subject can indicate that the subject has or ispredisposed to the development of a particular disease or condition.However, because these alleles are in linkage disequilibrium with otheralleles, the detection of such other linked alleles can also indicatethat the subject has or is predisposed to the development of aparticular disease or condition. For example, the 44112332 haplotypecomprises the following genotype: allele 4 of the 222/223 marker ofIL-1A allele 4 of the gz5/gz6 marker of IL-1A allele 1 of the −889marker of IL-1A allele 1 of the +3954 marker of IL-1B allele 2 of the−511 marker of IL-1B allele 3 of the gaat.p33330 marker allele 3 of theY31 marker allele 2 of +2018 of IL-1RN allele 1 of +4845 of IL-1A allele2 of the VNTR marker of IL-1RN

[0065] Three other polymorphisms in an IL-1RN alternative exon (Exonlic, which produces an intracellular form of the gene product) are alsoin linkage disequilibrium with allele 2 of IL-1RN (VNTR) (Clay et al.,(1996) Hum Genet 97:723-26). These include: IL-1RN exon lic (1812)(GenBank:X77090 at 1812); the IL-1RN exon lic (1868) polymorphism(GenBank:X77090 at 1868); and the IL-1RN exon lic (1887) polymorphism(GenBank:X77090 at 1887). Furthermore yet another polymorphism in thepromoter for the alternatively spliced intracellular form of the gene,the Pic (1731) polymorphism (GenBank:X77090 at 1731), is also in linkagedisequilibrium with allele 2 of the IL-1RN (VNTR) polymorphic locus. Foreach of these polymorphic loci, the allele 2 sequence variant has beendetermined to be in linkage disequilibrium with allele 2 of the IL-1RN(VNTR) locus (Clay et al., (1996) Hum Genet 97:723-26).

[0066] The 33221461 haplotype comprises the following genotype: allele 3of the 222/223 marker of IL-1A allele 3 of the gz5/gz6 marker of IL-1Aallele 2 of the −889 marker of IL-1A allele 2 of the +3954 marker ofIL-1B allele 1 of the −511 marker of IL-1B allele 4 of the gaat.p33330marker allele 6 of the Y31 marker allele 1 of +2018 of IL-1RN allele 2of +4845 of IL-1A allele 1 of the VNTR marker of IL-1RN

[0067] Individuals with the 44112332 haplotype are typicallyoverproducers of both IL-1α and IL-1β proteins, upon stimulation. Incontrast, individuals with the 33221461 haplotype are typicallyunderproducers of IL-1ra. Each haplotype results in a netproinflammatory response. Each allele within a haplotype may have aneffect, as well as a composite genotype effect. In addition, particulardiseases may be associated with both haplotype patterns.

[0068] The following Table 1 sets forth a number of genotype markers andvarious diseases and conditions to which these markers have been foundto be associated to a statistically significant extent. TABLE 1Association Of IL-1 Haplotype Gene Markers With Certain Diseases IL-1AIL-1A IL-1B IL-1B IL-1RN GENOTYPE (−889) (+4845) (−511) (+3954) (+2018)DISEASE Periodontal Disease (*2) *2 *2 Coronary Artery *2 *2 DiseaseAtherosclerosis Osteoporosis *2 Insulin dependent *2 diabetes Diabeticretinopathy *1 Endstage renal (+) diseases Diabetic nephropathy *2Hepatic fibrosis (+) (Japanese alcoholics) Alopecia areata *2 Graves'disease *2 Graves' (−) ophthalmopathy Extrathyroid disease (+) SystemicLupus *2 Erythematosus Lichen Sclerosis *2 Arthritis (+) Juvenilechronic *2 arthritis Rheumatoid arthritis (+) Insulin dependent *2 *2VNTR diabetes Ulcerative colitis *2 Asthma *2 *2 Multiple sclerosis (*2)*2VNTR Menopause, *2 early onset

[0069] In addition to the allelic patterns described above, as describedherein, one of skill in the art can readily identify other alleles(including polymorphisms and mutations) that are in linkagedisequilibrium with an allele associated with a disease or disorder. Forexample, a nucleic acid sample from a first group of subjects without aparticular disorder can be collected, as well as DNA from a second groupof subjects with the disorder. The nucleic acid sample can then becompared to identify those alleles that are over-represented in thesecond group as compared with the first group, wherein such alleles arepresumably associated with a disorder, which is caused or contributed toby inappropriate interleukin 1 regulation. Alternatively, alleles thatare in linkage disequilibrium with an allele that is associated with thedisorder can be identified, for example, by genotyping a largepopulation and performing statistical analysis to determine whichalleles appear more commonly together than expected. Preferably thegroup is chosen to be comprised of genetically related individuals.Genetically related individuals include individuals from the same race,the same ethnic group, or even the same family. As the degree of geneticrelatedness between a control group and a test group increases, so doesthe predictive value of polymorphic alleles which are ever moredistantly linked to a disease-causing allele. This is because lessevolutionary time has passed to allow polymorphisms which are linkedalong a chromosome in a founder population to redistribute throughgenetic cross-over events. Thus race-specific, ethnic-specific, and evenfamily-specific diagnostic genotyping assays can be developed to allowfor the detection of disease alleles which arose at ever more recenttimes in human evolution, e.g., after divergence of the major humanraces, after the separation of human populations into distinct ethnicgroups, and even within the recent history of a particular family line.

[0070] Linkage disequilibrium between two polymorphic markers or betweenone polymorphic marker and a disease-causing mutation is a meta-stablestate. Absent selective pressure or the sporadic linked reoccurrence ofthe underlying mutational events, the polymorphisms will eventuallybecome disassociated by chromosomal recombination events and willthereby reach linkage equilibrium through the course of human evolution.Thus, the likelihood of finding a polymorphic allele in linkagedisequilibrium with a disease or condition may increase with changes inat least two factors: decreasing physical distance between thepolymorphic marker and the disease-causing mutation, and decreasingnumber of meiotic generations available for the dissociation of thelinked pair. Consideration of the latter factor suggests that, the moreclosely related two individuals are, the more likely they will share acommon parental chromosome or chromosomal region containing the linkedpolymorphisms and the less likely that this linked pair will have becomeunlinked through meiotic cross-over events occurring each generation. Asa result, the more closely related two individuals are, the more likelyit is that widely spaced polymorphisms may be co-inherited. Thus, forindividuals related by common race, ethnicity or family, the reliabilityof ever more distantly spaced polymorphic loci can be relied upon as anindicator of inheritance of a linked disease-causing mutation.

[0071] Appropriate probes may be designed to hybridize to a specificgene of the IL-1 locus, such as IL-1A, IL-1B or IL-1RN or a relatedgene. These genomic DNA sequences are shown in FIGS. 3, 4 and 5,respectively, and further correspond to SEQ ID Nos. 1, 2 and 3,respectively. Alternatively, these probes may incorporate other regionsof the relevant genomic locus, including intergenic sequences. Indeedthe IL-1 region of human chromosome 2 spans some 400,000 base pairs and,assuming an average of one single nucleotide polymorphism every 1,000base pairs, includes some 400 SNPs loci alone. Yet other polymorphismsavailable for use with the immediate invention are obtainable fromvarious public sources. For example, the human genome database collectsintragenic SNPs, is searchable by sequence and currently containsapproximately 2,700 entries (http://hgbase.interactiva.de). Alsoavailable is a human polymorphism database maintained by theMassachusetts Institute of Technology (MIT SNP database(http://www.genome.wi.mit.edu/SNP/human/index.html)). From such sourcesSNPs as well as other human polymorphisms may be found.

[0072] For example, examination of the IL-1 region of the human genomein any one of these databases reveals that the IL-1 locus genes areflanked by a centromere proximal polymorphic marker designatedmicrosatellite marker AFM220ze3 at 127.4 cM (centiMorgans) (see GenBankAcc. No. Z17008) and a distal polymorphic marker designatedmicrosatellite anchor marker AFM087xa1 at 127.9 cM (see GenBank Acc. No.Z16545). These human polymorphic loci are both CA dinucleotide repeatmicrosatellite polymorphisms, and, as such, show a high degree ofheterozygosity in human populations. For example, one allele ofAFM220ze3 generates a 211 bp PCR amplification product with a 5′ primerof the sequence TGTACCTAAGCCCACCCTTTAGAGC (SEQ ID No. 4)

[0073] and a 3′ primer of the sequence TGGCCTCCAGAAACCTCCAA. (SEQ ID No.5)

[0074] Furthermore, one allele of AFM087xa1 generates a 177 bp PCRamplification product with a 5′ primer of the sequenceGCTGATATTCTGGTGGGAAA (SEQ ID No. 6)

[0075] and a 3′ primer of the sequence GGCAAGAGCAAAACTCTGTC. (SEQ ID No.7)

[0076] Equivalent primers corresponding to unique sequences occurring 5′and 3′ to these human chromosome 2 CA dinucleotide repeat polymorphismswill be apparent to one of skill in the art. Reasonable equivalentprimers include those which hybridize within about 1 kb of thedesignated primer, and which further are anywhere from about 17 bp toabout 27 bp in length. A general guideline for designing primers foramplification of unique human chromosomal genomic sequences is that theypossess a melting temperature of at least about 50^(B)C, wherein anapproximate melting temperature can be estimated using the formulaT_(melt)=[2×(# of A or T)+4×(# of G or C)].

[0077] A number of other human polymorphic loci occur between these twoCA dinucleotide repeat polymorphisms and provide additional targets fordetermination of a prognostic allele in a family or other group ofgenetically related individuals. For example, the National Center forBiotechnology Information web site (www.ncbi.nlm.nih.gov/genemap/) listsa number of polymorphism markers in the region of the IL-1 locus andprovides guidance in designing appropriate primers for amplification andanalysis of these markers.

[0078] Accordingly, the nucleotide segments of the invention may be usedfor their ability to selectively form duplex molecules withcomplementary stretches of human chromosome 2 q 12-13 or cDNAs from thatregion or to provide primers for amplification of DNA or cDNA from thisregion. The design of appropriate probes for this purpose requiresconsideration of a number of factors. For example, fragments having alength of between 10, 15, or 18 nucleotides to about 20, or to about 30nucleotides, will find particular utility. Longer sequences, e.g., 40,50, 80, 90, 100, even up to full length, are even more preferred forcertain embodiments. Lengths of oligonucleotides of at least about 18 to20 nucleotides are well accepted by those of skill in the art assufficient to allow sufficiently specific hybridization so as to beuseful as a molecular probe. Furthermore, depending on the applicationenvisioned, one will desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids. For example, relatively low salt and/or high temperatureconditions, such as provided by 0.02 M-0.15M NaCl at temperatures ofabout 50^(B)C to about 70^(B)C. Such selective conditions may toleratelittle, if any, mismatch between the probe and the template or targetstrand.

[0079] Other alleles or other indicia of a disorder can be detected ormonitored in a subject in conjunction with detection of the allelesdescribed above, for example, identifying vessel wall thickness (e.g. asmeasured by ultrasound), or whether the subject smokes, drinks isoverweight, is under stress or exercises.

4.2.2 Detection of Alleles

[0080] Many methods are available for detecting specific alleles athuman polymorphic loci. The preferred method for detecting a specificpolymorphic allele will depend, in part, upon the molecular nature ofthe polymorphism. For example, the various allelic forms of thepolymorphic locus may differ by a single base-pair of the DNA. Suchsingle nucleotide polymorphisms (or SNPs) are major contributors togenetic variation, comprising some 80% of all known polymorphisms, andtheir density in the human genome is estimated to be on average 1 per1,000 base pairs. SNPs are most frequently biallelic—occurring in onlytwo different forms (although up to four different forms of an SNP,corresponding to the four different nucleotide bases occurring in DNA,are theoretically possible). Nevertheless, SNPs are mutationally morestable than other polymorphisms, making them suitable for associationstudies in which linkage disequilibrium between markers and an unknownvariant is used to map disease-causing mutations. In addition, becauseSNPs typically have only two alleles, they can be genotyped by a simpleplus/minus assay rather than a length measurement, making them moreamenable to automation.

[0081] A variety of methods are available for detecting the presence ofa particular single nucleotide polymorphic allele in an individual.Advancements in this field have provided accurate, easy, and inexpensivelarge-scale SNP genotyping. Most recently, for example, several newtechniques have been described including dynamic allele-specifichybridization (DASH), microplate array diagonal gel electrophoresis(MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMansystem as well as various DNA “chip” technologies such as the AffymetrixSNP chips. These methods require amplification of the target geneticregion, typically by PCR. Still other newly developed methods, based onthe generation of small signal molecules by invasive cleavage followedby mass spectrometry or immobilized padlock probes and rolling-circleamplification, might eventually eliminate the need for PCR. Several ofthe methods known in the art for detecting specific single nucleotidepolymorphisms are summarized below. The method of the present inventionis understood to include all available methods.

[0082] Several methods have been developed to facilitate analysis ofsingle nucleotide polymorphisms. In one embodiment, the single basepolymorphism can be detected by using a specializedexonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C. R.(U.S. Pat. No. 4,656,127). According to the method, a primercomplementary to the allelic sequence immediately 3′ to the polymorphicsite is permitted to hybridize to a target molecule obtained from aparticular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

[0083] In another embodiment of the invention, a solution-based methodis used for determining the identity of the nucleotide of a polymorphicsite. Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No.WO91/02087). As in the Mundy method of U.S. Pat. No. 4,656,127, a primeris employed that is complementary to allelic sequences immediately 3′ toa polymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

[0084] An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

[0085] Recently, several primer-guided nucleotide incorporationprocedures for assaying polymorphic sites in DNA have been described(Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov,B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A. -C., et al.,Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl. Acad.Sci. (U.S.A.) 88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat.1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P.et al., Anal. Biochem. 208:171-175 (1993)). These methods differ fromGBA™ in that they all rely on the incorporation of labeleddeoxynucleotides to discriminate between bases at a polymorphic site. Insuch a format, since the signal is proportional to the number ofdeoxynucleotides incorporated, polymorphisms that occur in runs of thesame nucleotide can result in signals that are proportional to thelength of the run (Syvanen, A. -C., et al., Amer. J. Hum. Genet.52:46-59 (1993)).

[0086] For mutations that produce premature termination of proteintranslation, the protein truncation test (PTT) offers an efficientdiagnostic approach (Roest, et. al., (1993) Hum. Mol. Genet. 2:1719-21;van der Luijt, et. al., (1994) Genomics 20:1-4). For PTT, RNA isinitially isolated from available tissue and reverse-transcribed, andthe segment of interest is amplified by PCR. The products of reversetranscription PCR are then used as a template for nested PCRamplification with a primer that contains an RNA polymerase promoter anda sequence for initiating eukaryotic translation. After amplification ofthe region of interest, the unique motifs incorporated into the primerpermit sequential in vitro transcription and translation of the PCRproducts. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresisof translation products, the appearance of truncated polypeptidessignals the presence of a mutation that causes premature termination oftranslation. In a variation of this technique, DNA (as opposed to RNA)is used as a PCR template when the target region of interest is derivedfrom a single exon.

[0087] Any cell type or tissue may be utilized to obtain nucleic acidsamples for use in the diagnostics described herein. In a preferredembodiment, the DNA sample is obtained from a bodily fluid, e.g, blood,obtained by known techniques (e.g. venipuncture) or saliva.Alternatively, nucleic acid tests can be performed on dry samples (e.g.hair or skin). When using RNA or protein, the cells or tissues that maybe utilized must express an IL-1 gene.

[0088] Diagnostic procedures may also be performed in situ directly upontissue sections (fixed and/or frozen) of patient tissue obtained frombiopsies or resections, such that no nucleic acid purification isnecessary. Nucleic acid reagents may be used as probes and/or primersfor such in situ procedures (see, for example, Nuovo, G. J., 1992, PCRin situ hybridization: protocols and applications, Raven Press, NY).

[0089] In addition to methods which focus primarily on the detection ofone nucleic acid sequence, profiles may also be assessed in suchdetection schemes. Fingerprint profiles may be generated, for example,by utilizing a differential display procedure, Northern analysis and/orRT-PCR.

[0090] A preferred detection method is allele specific hybridizationusing probes overlapping a region of at least one allele of an IL-1proinflammatory haplotype and having about 5, 10, 20, 25, or 30nucleotides around the mutation or polymorphic region. In a preferredembodiment of the invention, several probes capable of hybridizingspecifically to other allelic variants involved in a restenosis areattached to a solid phase support, e.g., a “chip” (which can hold up toabout 250,000 oligonucleotides). Oligonucleotides can be bound to asolid support by a variety of processes, including lithography. Mutationdetection analysis using these chips comprising oligonucleotides, alsotermed “DNA probe arrays” is described e.g., in Cronin et al. (1996)Human Mutation 7:244. In one embodiment, a chip comprises all theallelic variants of at least one polymorphic region of a gene. The solidphase support is then contacted with a test nucleic acid andhybridization to the specific probes is detected. Accordingly, theidentity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment.

[0091] These techniques may also comprise the step of amplifying thenucleic acid before analysis. Amplification techniques are known tothose of skill in the art and include, but are not limited to cloning,polymerase chain reaction (PCR), polymerase chain reaction of specificalleles (ASA), ligase chain reaction (LCR), nested polymerase chainreaction, self sustained sequence replication (Guatelli, J. C. et al.,1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptionalamplification system (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci.USA 86:1173-1177), and Q- Beta Replicase (Lizardi, P. M. et al., 1988,Bio/Technology 6:1197).

[0092] Amplification products may be assayed in a variety of ways,including size analysis, restriction digestion followed by sizeanalysis, detecting specific tagged oligonucleotide primers in thereaction products, allele-specific oligonucleotide (ASO) hybridization,allele specific 5′ exonuclease detection, sequencing, hybridization, andthe like.

[0093] PCR based detection means can include multiplex amplification ofa plurality of markers simultaneously. For example, it is well known inthe art to select PCR primers to generate PCR products that do notoverlap in size and can be analyzed simultaneously. Alternatively, it ispossible to amplify different markers with primers that aredifferentially labeled and thus can each be differentially detected. Ofcourse, hybridization based detection means allow the differentialdetection of multiple PCR products in a sample. Other techniques areknown in the art to allow multiplex analyses of a plurality of markers.

[0094] In a merely illustrative embodiment, the method includes thesteps of (i) collecting a sample of cells from a patient, (ii) isolatingnucleic acid (e.g., genomic, mRNA or both) from the cells of the sample,(iii) contacting the nucleic acid sample with one or more primers whichspecifically hybridize 5′ and 3′ to at least one allele of an IL-1proinflammatory haplotype under conditions such that hybridization andamplification of the allele occurs, and (iv) detecting the amplificationproduct. These detection schemes are especially useful for the detectionof nucleic acid molecules if such molecules are present in very lownumbers.

[0095] In a preferred embodiment of the subject assay, the allele of anIL-1 proinflammatory haplotype is identified by alterations inrestriction enzyme cleavage patterns. For example, sample and controlDNA is isolated, amplified (optionally), digested with one or morerestriction endonucleases, and fragment length sizes are determined bygel electrophoresis.

[0096] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the allele.Exemplary sequencing reactions include those based on techniquesdeveloped by Maxim and Gilbert ((1977) Proc. Natl Acad Sci USA 74:560)or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci USA 74:5463). It isalso contemplated that any of a variety of automated sequencingprocedures may be utilized when performing the subject assays (see, forexample Biotechniques (1995) 19:448), including sequencing by massspectrometry (see, for example PCT publication WO 94/16101; Cohen et al.(1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl BiochemBiotechnol 38:147-159). It will be evident to one of skill in the artthat, for certain embodiments, the occurrence of only one, two or threeof the nucleic acid bases need be determined in the sequencing reaction.For instance, A-track or the like, e.g., where only one nucleic acid isdetected, can be carried out.

[0097] In a further embodiment, protection from cleavage agents (such asa nuclease, hydroxylamine or osmium tetroxide and with piperidine) canbe used to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, theart technique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing (labeled) RNA or DNA containing the wild-typeallele with the sample. The double-stranded duplexes are treated with anagent which cleaves single-stranded regions of the duplex such as whichwill exist due to base pair mismatches between the control and samplestrands. For instance, RNA/DNA duplexes can be treated with RNase andDNA/DNA hybrids treated with S1 nuclease to enzymatically digest themismatched regions. In other embodiments, either DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions, the resulting material is then separated by size ondenaturing polyacrylamide gels to determine the site of mutation. See,for example, Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397; andSaleeba et al (1992) Methods Enzymol. 217:286-295. In a preferredembodiment, the control DNA or RNA can be labeled for detection.

[0098] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes). Forexample, the mutY enzyme of E. coli cleaves A at G/A mismatches and thethymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches(Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to anexemplary embodiment, a probe based on an allele of an IL-1 locushaplotype is hybridized to a cDNA or other DNA product from a testcell(s). The duplex is treated with a DNA mismatch repair enzyme, andthe cleavage products, if any, can be detected from electrophoresisprotocols or the like. See, for example, U.S. Pat. No. 5,459,039.

[0099] In other embodiments, alterations in electrophoretic mobilitywill be used to identify an IL-1 locus allele. For example, singlestrand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766, seealso Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet AnalTech Appl 9:73-79). Single-stranded DNA fragments of sample and controlIL-1 locus alleles are denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[0100] In yet another embodiment, the movement of alleles inpolyacrylamide gels containing a gradient of denaturant is assayed usingdenaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985)Nature 313:495). When DGGE is used as the method of analysis, DNA willbe modified to insure that it does not completely denature, for exampleby adding a GC clamp of approximately 40 bp of high-melting GC-rich DNAby PCR. In a further embodiment, a temperature gradient is used in placeof a denaturing agent gradient to identify differences in the mobilityof control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem265:12753).

[0101] Examples of other techniques for detecting alleles include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation ornucleotide difference (e.g., in allelic variants) is placed centrallyand then hybridized to target DNA under conditions which permithybridization only if a perfect match is found (Saiki et al. (1986)Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230).Such allele specific oligonucleotide hybridization techniques may beused to test one mutation or polymorphic region per reaction whenoligonucleotides are hybridized to PCR amplified target DNA or a numberof different mutations or polymorphic regions when the oligonucleotidesare attached to the hybridizing membrane and hybridized with labelledtarget DNA.

[0102] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation or polymorphic region of interestin the center of the molecule (so that amplification depends ondifferential hybridization) (Gibbs et al (1989) Nucleic Acids Res.17:2437-2448) or at the extreme 3′ end of one primer where, underappropriate conditions, mismatch can prevent, or reduce polymeraseextension (Prossner (1993) Tibtech 11:238. In addition it may bedesirable to introduce a novel restriction site in the region of themutation to create cleavage-based detection (Gasparini et al (1992) Mol.Cell Probes 6:1). It is anticipated that in certain embodimentsamplification may also be performed using Taq ligase for amplification(Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases,ligation will occur only if there is a perfect match at the 3′ end ofthe 5′ sequence making it possible to detect the presence of a knownmutation at a specific site by looking for the presence or absence ofamplification.

[0103] In another embodiment, identification of the allelic variant iscarried out using an oligonucleotide ligation assay (OLA), as described,e.g., in U.S. Pat. No. 4,998,617 and in Landegren, U. et al. ((1988)Science 241:1077-1080). The OLA protocol uses two oligonucleotides whichare designed to be capable of hybridizing to abutting sequences of asingle strand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al. (1990)Proc. Natl. Acad. Sci. USA 87:8923-27). In this method, PCR is used toachieve the exponential amplification of target DNA, which is thendetected using OLA.

[0104] Several techniques based on this OLA method have been developedand can be used to detect alleles of an IL-1 locus haplotype. Forexample, U.S. Pat. No. 5,593,826 discloses an OLA using anoligonucleotide having 3′-amino group and a 5′-phosphorylatedoligonucleotide to form a conjugate having a phosphoramidate linkage. Inanother variation of OLA described in Tobe et al. ((1996) Nucleic AcidsRes 24: 3728), OLA combined with PCR permits typing of two alleles in asingle microtiter well. By marking each of the allele-specific primerswith a unique hapten, i.e. digoxigenin and fluorescein, each OLAreaction can be detected by using hapten specific antibodies that arelabeled with different enzyme reporters, alkaline phosphatase orhorseradish peroxidase. This system permits the detection of the twoalleles using a high throughput format that leads to the production oftwo different colors.

[0105] Another embodiment of the invention is directed to kits fordetecting a predisposition for developing a restenosis. This kit maycontain one or more oligonucleotides, including 5′ and 3′oligonucleotides that hybridize 5′ and 3′ to at least one allele of anIL-1 locus haplotype. PCR amplification oligonucleotides shouldhybridize between 25 and 2500 base pairs apart, preferably between about100 and about 500 bases apart, in order to produce a PCR product ofconvenient size for subsequent analysis.

[0106] Particularly preferred primers for use in the diagnostic methodof the invention include SEQ ID Nos. 1-6

[0107] The design of additional oligonucleotides for use in theamplification and detection of IL-1 polymorphic alleles by the method ofthe invention is facilitated by the availability of both updatedsequence information from human chromosome 2q13—which contains the humanIL-1 locus, and updated human polymorphism information available forthis locus. For example, the DNA sequence for the IL-1A, IL-1B andIL-1RN is shown in FIGS. 1 (GenBank Accession No. X03833), 2 (GenBankAccession No. X04500) and 3 (GenBank Accession No. X64532) respectively.Suitable primers for the detection of a human polymorphism in thesegenes can be readily designed using this sequence information andstandard techniques known in the art for the design and optimization ofprimers sequences. Optimal design of such primer sequences can beachieved, for example, by the use of commercially available primerselection programs such as Primer 2.1, Primer 3 or GeneFisher (See also,Nicklin M. H. J., Weith A. Duff G. W., “A Physical Map of the RegionEncompassing the Human Interleukin-1α, interleukin-1β, and Interleukin-1Receptor Antagonist Genes” Genomics 19: 382 (1995); Nothwang H. G., etal. “Molecular Cloning of the Interleukin-1 gene Cluster: Constructionof an Integrated YAC/PAC Contig and a partial transcriptional Map in theRegion of Chromosome 2q13” Genomics 41: 370 (1997); Clark, et al. (1986)Nucl. Acids. Res., 14:7897-7914 [published erratum appears in NucleicAcids Res., 15:868 (1987) and the Genome Database (GDB) project at theURL http://www.gdb.org).

[0108] For use in a kit, oligonucleotides may be any of a variety ofnatural and/or synthetic compositions such as syntheticoligonucleotides, restriction fragments, cDNAs, synthetic peptidenucleic acids (PNAs), and the like. The assay kit and method may alsoemploy labeled oligonucleotides to allow ease of identification in theassays. Examples of labels which may be employed include radio-labels,enzymes, fluorescent compounds, streptavidin, avidin, biotin, magneticmoieties, metal binding moieties, antigen or antibody moieties, and thelike.

[0109] The kit may, optionally, also include DNA sampling means. DNAsampling means are well known to one of skill in the art and caninclude, but not be limited to substrates, such as filter papers, theAmpliCard™ (University of Sheffield, Sheffield, England S10 2JF; Tarlow,J W, et al., J of Invest. Dermatol. 103:387-389 (1994)) and the like;DNA purification reagents such as Nucleon™ kits, lysis buffers,proteinase solutions and the like; PCR reagents, such as 10× reactionbuffers, thermostable polymerase, dNTPs, and the like; and alleledetection means such as the HinfI restriction enzyme, allele specificoligonucleotides, degenerate oligonucleotide primers for nested PCR fromdried blood.

4.2.3. Pharmacogenomics

[0110] Knowledge of the particular alleles associated with asusceptibility to developing a particular disease or condition, alone orin conjunction with information on other genetic defects contributing tothe particular disease or condition allows a customization of theprevention or treatment in accordance with the individual's geneticprofile, the goal of “pharmacogenomics”. Thus, comparison of anindividual's IL-1 profile to the population profile for a vasculardisorder, permits the selection or design of drugs or other therapeuticregimens that are expected to be safe and efficacious for a particularpatient or patient population (i.e., a group of patients having the samegenetic alteration).

[0111] In addition, the ability to target populations expected to showthe highest clinical benefit, based on genetic profile can enable: 1)the repositioning of already marketed drugs; 2) the rescue of drugcandidates whose clinical development has been discontinued as a resultof safety or efficacy limitations, which are patient subgroup-specific;and 3) an accelerated and less costly development for candidatetherapeutics and more optimal drug labeling (e.g. since measuring theeffect of various doses of an agent on the causative mutation is usefulfor optimizing effective dose).

[0112] The treatment of an individual with a particular therapeutic canbe monitored by determining protein (e.g. IL-1α, IL-1β, or IL-1Ra), mRNAand/or transcriptional level. Depending on the level detected, thetherapeutic regimen can then be maintained or adjusted (increased ordecreased in dose). In a preferred embodiment, the effectiveness oftreating a subject with an agent comprises the steps of: (i) obtaining apreadministration sample from a subject prior to administration of theagent; (ii) detecting the level or amount of a protein, mRNA or genomicDNA in the preadministration sample; (iii) obtaining one or morepost-administration samples from the subject; (iv) detecting the levelof expression or activity of the protein, mRNA or genomic DNA in thepost-administration sample; (v) comparing the level of expression oractivity of the protein, mRNA or genomic DNA in the preadministrationsample with the corresponding protein, mRNA or genomic DNA in thepostadministration sample, respectively; and (vi) altering theadministration of the agent to the subject accordingly.

[0113] Cells of a subject may also be obtained before and afteradministration of a therapeutic to detect the level of expression ofgenes other than an IL-1 gene to verify that the therapeutic does notincrease or decrease the expression of genes which could be deleterious.This can be done, e.g., by using the method of transcriptionalprofiling. Thus, mRNA from cells exposed in vivo to a therapeutic andmRNA from the same type of cells that were not exposed to thetherapeutic could be reverse transcribed and hybridized to a chipcontaining DNA from numerous genes, to thereby compare the expression ofgenes in cells treated and not treated with the therapeutic.

4.3 Therapeutics For Diseases and Conditions Associated with IL-1Polymorphisms

[0114] Therapeutic for diseases or conditions associated with an IL-1polymorphism or haplotype refers to any agent or therapeutic regimen(including pharmaceuticals, nutraceuticals and surgical means) thatprevents or postpones the development of or alleviates the symptoms ofthe particular disease or condition in the subject. The therapeutic canbe a polypeptide, peptidomimetic, nucleic acid or other inorganic ororganic molecule, preferably a “small molecule” including vitamins,minerals and other nutrients. Preferably the therapeutic can modulate atleast one activity of an IL-1 polypeptide, e.g., interaction with areceptor, by mimicking or potentiating (agonizing) or inhibiting(antagonizing) the effects of a naturally-occurring polypeptide. Anagonist can be a wild-type protein or derivative thereof having at leastone bioactivity of the wild-type, e.g., receptor binding activity. Anagonist can also be a compound that upregulates expression of a gene orwhich increases at least one bioactivity of a protein. An agonist canalso be a compound which increases the interaction of a polypeptide withanother molecule, e.g., a receptor. An antagonist can be a compoundwhich inhibits or decreases the interaction between a protein andanother molecule, e.g., a receptor or an agent that blocks signaltransduction or post-translation processing (e.g., IL-1 convertingenzyme (ICE) inhibitor). Accordingly, a preferred antagonist is acompound which inhibits or decreases binding to a receptor and therebyblocks subsequent activation of the receptor. An antagonist can also bea compound that downregulates expression of a gene or which reduces theamount of a protein present. The antagonist can be a dominant negativeform of a polypeptide, e.g., a form of a polypeptide which is capable ofinteracting with a target peptide, e.g., a receptor, but which does notpromote the activation of the receptor. The antagonist can also be anucleic acid encoding a dominant negative form of a polypeptide, anantisense nucleic acid, or a ribozyme capable of interactingspecifically with an RNA. Yet other antagonists are molecules which bindto a polypeptide and inhibit its action. Such molecules includepeptides, e.g., forms of target peptides which do not have biologicalactivity, and which inhibit binding to receptors. Thus, such peptideswill bind to the active site of a protein and prevent it frominteracting with target peptides. Yet other antagonists includeantibodies that specifically interact with an epitope of a molecule,such that binding interferes with the biological function of thepolypeptide. In yet another preferred embodiment, the antagonist is asmall molecule, such as a molecule capable of inhibiting the interactionbetween a polypeptide and a target receptor. Alternatively, the smallmolecule can function as an antagonist by interacting with sites otherthan the receptor binding site.

[0115] Modulators of IL-1 (e.g. IL-1αa, IL-1β or IL-1 receptorantagonist) or a protein encoded by a gene that is in linkagedisequilibrium with an IL-1 gene can comprise any type of compound,including a protein, peptide, peptidomimetic, small molecule, or nucleicacid. Preferred agonists include nucleic acids (e.g. encoding an IL-1protein or a gene that is up- or down-regulated by an IL-1 protein),proteins (e.g. IL-1 proteins or a protein that is up- or down-regulatedthereby) or a small molecule (e.g. that regulates expression or bindingof an IL-1 protein). Preferred antagonists, which can be identified, forexample, using the assays described herein, include nucleic acids (e.g.single (antisense) or double stranded (triplex) DNA or PNA andribozymes), protein (e.g. antibodies) and small molecules that act tosuppress or inhibit IL-1 transcription and/or protein activity.

4.3.1. Effective Dose

[0116] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining The LD50 (the dose lethal to50% of the population) and the Ed50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissues in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0117] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

4.3.2. Formulation and Use

[0118] Compositions for use in accordance with the present invention maybe formulated in a conventional manner using one or more physiologicallyacceptable carriers or excipients. Thus, the compounds and theirphysiologically acceptable salts and solvates may be formulated foradministration by, for example, injection, inhalation or insufflation(either through the mouth or the nose) or oral, buccal, parenteral orrectal administration.

[0119] For such therapy, the compounds of the invention can beformulated for a variety of loads of administration, including systemicand topical or localized administration. Techniques and formulationsgenerally may be found in Remmington's Pharmaceutical Sciences, MeadePublishing Co., Easton, Pa. For systemic administration, injection ispreferred, including intramuscular, intravenous, intraperitoneal, andsubcutaneous. For injection, the compounds of the invention can beformulated in liquid solutions, preferably in physiologically compatiblebuffers such as Hank's solution or Ringer's solution. In addition, thecompounds may be formulated in solid form and redissolved or suspendedimmediately prior to use. Lyophilized forms are also included.

[0120] For oral administration, the compositions may take the form of,for example, tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulfate). The tabletsmay be coated by methods well known in the art. Liquid preparations fororal administration may take the form of, for example, solutions, syrupsor suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

[0121] Preparations for oral administration may be suitably formulatedto give controlled release of the active compound. For buccaladministration the compositions may take the form of tablets or lozengesformulated in conventional manner. For administration by inhalation, thecompounds for use according to the present invention are convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

[0122] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulating agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0123] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0124] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt. Other suitable delivery systemsinclude microspheres which offer the possibility of local noninvasivedelivery of drugs over an extended period of time. This technologyutilizes microspheres of precapillary size which can be injected via acoronary catheter into any selected part of the e.g. heart or otherorgans without causing inflammation or ischemia. The administeredtherapeutic is slowly released from these microspheres and taken up bysurrounding tissue cells (e.g. endothelial cells).

[0125] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration bile salts andfusidic acid derivatives. In addition, detergents may be used tofacilitate permeation. Transmucosal administration may be through nasalsprays or using suppositories. For topical administration, the oligomersof the invention are formulated into ointments, salves, gels, or creamsas generally known in the art. A wash solution can be used locally totreat an injury or inflammation to accelerate healing.

[0126] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

4.4 Assays to Identify Therapeutics

[0127] Based on the identification of mutations that cause or contributeto the development of a disease or disorder that is associated with anIL-1 polymorphism or haplotype, the invention further featurescell-based or cell free assays for identifying therapeutics. In oneembodiment, a cell expressing an IL-1 receptor, or a receptor for aprotein that is encoded by a gene which is in linkage disequilibriumwith an IL-1 gene, on the outer surface of its cellular membrane isincubated in the presence of a test compound alone or in the presence ofa test compound and another protein and the interaction between the testcompound and the receptor or between the protein (preferably a taggedprotein) and the receptor is detected, e.g., by using a microphysiometer(McConnell et al. (1992) Science 257:1906). An interaction between thereceptor and either the test compound or the protein is detected by themicrophysiometer as a change in the acidification of the medium. Thisassay system thus provides a means of identifying molecular antagonistswhich, for example, function by interfering with protein-receptorinteractions, as well as molecular agonist which, for example, functionby activating a receptor.

[0128] Cellular or cell-free assays can also be used to identifycompounds which modulate expression of an IL-1 gene or a gene in linkagedisequilibrium therewith, modulate translation of an mRNA, or whichmodulate the stability of an mRNA or protein. Accordingly, in oneembodiment, a cell which is capable of producing an IL-1, or otherprotein is incubated with a test compound and the amount of proteinproduced in the cell medium is measured and compared to that producedfrom a cell which has not been contacted with the test compound. Thespecificity of the compound vis a vis the protein can be confirmed byvarious control analysis, e.g., measuring the expression of one or morecontrol genes. In particular, this assay can be used to determine theefficacy of antisense, ribozyme and triplex compounds.

[0129] Cell-free assays can also be used to identify compounds which arecapable of interacting with a protein, to thereby modify the activity ofthe protein. Such a compound can, e.g., modify the structure of aprotein thereby effecting its ability to bind to a receptor. In apreferred embodiment, cell-free assays for identifying such compoundsconsist essentially in a reaction mixture containing a protein and atest compound or a library of test compounds in the presence or absenceof a binding partner. A test compound can be, e.g., a derivative of abinding partner, e.g., a biologically inactive target peptide, or asmall molecule.

[0130] Accordingly, one exemplary screening assay of the presentinvention includes the steps of contacting a protein or functionalfragment thereof with a test compound or library of test compounds anddetecting the formation of complexes. For detection purposes, themolecule can be labeled with a specific marker and the test compound orlibrary of test compounds labeled with a different marker. Interactionof a test compound with a protein or fragment thereof can then bedetected by determining the level of the two labels after an incubationstep and a washing step. The presence of two labels after the washingstep is indicative of an interaction.

[0131] An interaction between molecules can also be identified by usingreal-time BIA (Biomolecular Interaction Analysis, Pharmacia BiosensorAB) which detects surface plasmon resonance (SPR), an opticalphenomenon. Detection depends on changes in the mass concentration ofmacromolecules at the biospecific interface, and does not require anylabeling of interactants. In one embodiment, a library of test compoundscan be immobilized on a sensor surface, e.g., which forms one wall of amicro-flow cell. A solution containing the protein or functionalfragment thereof is then flown continuously over the sensor surface. Achange in the resonance angle as shown on a signal recording, indicatesthat an interaction has occurred. This technique is further described,e.g., in BIAtechnology Handbook by Pharmacia.

[0132] Another exemplary screening assay of the present inventionincludes the steps of (a) forming a reaction mixture including: (i) anIL-1 or other protein, (ii) an appropriate receptor, and (iii) a testcompound; and (b) detecting interaction of the protein and receptor. Astatistically significant change (potentiation or inhibition) in theinteraction of the protein and receptor in the presence of the testcompound, relative to the interaction in the absence of the testcompound, indicates a potential antagonist (inhibitor). The compounds ofthis assay can be contacted simultaneously. Alternatively, a protein canfirst be contacted with a test compound for an appropriate amount oftime, following which the receptor is added to the reaction mixture. Theefficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison.

[0133] Complex formation between a protein and receptor may be detectedby a variety of techniques. Modulation of the formation of complexes canbe quantitated using, for example, detectably labeled proteins such asradiolabeled, fluorescently labeled, or enzymatically labeled proteinsor receptors, by immunoassay, or by chromatographic detection.

[0134] Typically, it will be desirable to immobilize either the proteinor the receptor to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of protein and receptor can beaccomplished in any vessel suitable for containing the reactants.Examples include microtitre plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows the protein to be bound to a matrix. For example,glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe receptor, e.g. an 35S-labeled receptor, and the test compound, andthe mixture incubated under conditions conducive to complex formation,e.g. at physiological conditions for salt and pH, though slightly morestringent conditions may be desired. Following incubation, the beads arewashed to remove any unbound label, and the matrix immobilized andradiolabel determined directly (e.g. beads placed in scintillant), or inthe supernatant after the complexes are subsequently dissociated.Alternatively, the complexes can be dissociated from the matrix,separated by SDS-PAGE, and the level of protein or receptor found in thebead fraction quantitated from the gel using standard electrophoretictechniques such as described in the appended examples. Other techniquesfor immobilizing proteins on matrices are also available for use in thesubject assay. For instance, either protein or receptor can beimmobilized utilizing conjugation of biotin and streptavidin. Transgenicanimals can also be made to identify agonists and antagonists or toconfirm the safety and efficacy of a candidate therapeutic. Transgenicanimals of the invention can include non-human animals containing arestenosis causative mutation under the control of an appropriateendogenous promoter or under the control of a heterologous promoter.

[0135] The transgenic animals can also be animals containing atransgene, such as reporter gene, under the control of an appropriatepromoter or fragment thereof. These animals are useful, e.g., foridentifying drugs that modulate production of an IL-1 protein, such asby modulating gene expression. Methods for obtaining transgenicnon-human animals are well known in the art. In preferred embodiments,the expression of the restenosis causative mutation is restricted tospecific subsets of cells, tissues or developmental stages utilizing,for example, cis-acting sequences that control expression in the desiredpattern. In the present invention, such mosaic expression of a proteincan be essential for many forms of lineage analysis and can additionallyprovide a means to assess the effects of, for example, expression levelwhich might grossly alter development in small patches of tissue withinan otherwise normal embryo. Toward this end, tissue-specific regulatorysequences and conditional regulatory sequences can be used to controlexpression of the mutation in certain spatial patterns. Moreover,temporal patterns of expression can be provided by, for example,conditional recombination systems or prokaryotic transcriptionalregulatory sequences. Genetic techniques, which allow for the expressionof a mutation can be regulated via site-specific genetic manipulation invivo, are known to those skilled in the art.

[0136] The transgenic animals of the present invention all includewithin a plurality of their cells a causative mutation transgene of thepresent invention, which transgene alters the phenotype of the “hostcell”. In an illustrative embodiment, either the cre/loxP recombinasesystem of bacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orbanet al. (1992) PNAS 89:6861-6865) or the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355;PCT publication WO 92/15694) can be used to generate in vivosite-specific genetic recombination systems. Cre recombinase catalyzesthe site-specific recombination of an intervening target sequencelocated between loxP sequences. loxP sequences are 34 base pairnucleotide repeat sequences to which the Cre recombinase binds and arerequired for Cre recombinase mediated genetic recombination. Theorientation of loxP sequences determines whether the intervening targetsequence is excised or inverted when Cre recombinase is present(Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing theexcision of the target sequence when the loxP sequences are oriented asdirect repeats and catalyzes inversion of the target sequence when loxPsequences are oriented as inverted repeats.

[0137] Accordingly, genetic recombination of the target sequence isdependent on expression of the Cre recombinase. Expression of therecombinase can be regulated by promoter elements which are subject toregulatory control, e.g., tissue-specific, developmental stage-specific,inducible or repressible by externally added agents. This regulatedcontrol will result in genetic recombination of the target sequence onlyin cells where recombinase expression is mediated by the promoterelement. Thus, the activation of expression of the causative mutationtransgene can be regulated via control of recombinase expression.

[0138] Use of the cre/loxP recombinase system to regulate expression ofa causative mutation transgene requires the construction of a transgenicanimal containing transgenes encoding both the Cre recombinase and thesubject protein. Animals containing both the Cre recombinase and therestenosis causative mutation transgene can be provided through theconstruction of “double” transgenic animals. A convenient method forproviding such animals is to mate two transgenic animals each containinga transgene.

[0139] Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the transgene. Exemplarypromoters and the corresponding trans-activating prokaryotic proteinsare given in U.S. Pat. No. 4,833,080.

[0140] Moreover, expression of the conditional transgenes can be inducedby gene therapy-like methods wherein a gene encoding the transactivatingprotein, e.g. a recombinase or a prokaryotic protein, is delivered tothe tissue and caused to be expressed, such as in a cell-type specificmanner. By this method, the transgene could remain silent into adulthooduntil “turned on” by the introduction of the transactivator.

[0141] In an exemplary embodiment, the “transgenic non-human animals” ofthe invention are produced by introducing transgenes into the germlineof the non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thespecific line(s) of any animal used to practice this invention areselected for general good health, good embryo yields, good pronuclearvisibility in the embryo, and good reproductive fitness. In addition,the haplotype is a significant factor. For example, when transgenic miceare to be produced, strains such as C57BL/6 or FVB lines are often used(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those withH-2b, H-2d or H-2q haplotypes such as C57BL/6 or DBA/1. The line(s) usedto practice this invention may themselves be transgenics, and/or may beknockouts (i.e., obtained from animals which have one or more genespartially or completely suppressed).

[0142] In one embodiment, the transgene construct is introduced into asingle stage embryo. The zygote is the best target for microinjection.In the mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter which allows reproducible injection of 1-2 pl ofDNA solution. The use of zygotes as a target for gene transfer has amajor advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal. (1985) PNAS 82:4438-4442). As a consequence, all cells of thetransgenic animal will carry the incorporated transgene. This will ingeneral also be reflected in the efficient transmission of the transgeneto offspring of the founder since 50% of the germ cells will harbor thetransgene.

[0143] Normally, fertilized embryos are incubated in suitable mediauntil the pronuclei appear. At about this time, the nucleotide sequencecomprising the transgene is introduced into the female or malepronucleus as described below. In some species such as mice, the malepronucleus is preferred. It is most preferred that the exogenous geneticmaterial be added to the male DNA complement of the zygote prior to itsbeing processed by the ovum nucleus or the zygote female pronucleus. Itis thought that the ovum nucleus or female pronucleus release moleculeswhich affect the male DNA complement, perhaps by replacing theprotamines of the male DNA with histones, thereby facilitating thecombination of the female and male DNA complements to form the diploidzygote. Thus, it is preferred that the exogenous genetic material beadded to the male complement of DNA or any other complement of DNA priorto its being affected by the female pronucleus. For example, theexogenous genetic material is added to the early male pronucleus, assoon as possible after the formation of the male pronucleus, which iswhen the male and female pronuclei are well separated and both arelocated close to the cell membrane. Alternatively, the exogenous geneticmaterial could be added to the nucleus of the sperm after it has beeninduced to undergo decondensation. Sperm containing the exogenousgenetic material can then be added to the ovum or the decondensed spermcould be added to the ovum with the transgene constructs being added assoon as possible thereafter.

[0144] Introduction of the transgene nucleotide sequence into the embryomay be accomplished by any means known in the art such as, for example,microinjection, electroporation, or lipofection. Following introductionof the transgene nucleotide sequence into the embryo, the embryo may beincubated in vitro for varying amounts of time, or reimplanted into thesurrogate host, or both. In vitro incubation to maturity is within thescope of this invention. One common method in to incubate the embryos invitro for about 1-7 days, depending on the species, and then reimplantthem into the surrogate host.

[0145] For the purposes of this invention a zygote is essentially theformation of a diploid cell which is capable of developing into acomplete organism. Generally, the zygote will be comprised of an eggcontaining a nucleus formed, either naturally or artificially, by thefusion of two haploid nuclei from a gamete or gametes. Thus, the gametenuclei must be ones which are naturally compatible, i.e., ones whichresult in a viable zygote capable of undergoing differentiation anddeveloping into a functioning organism. Generally, a euploid zygote ispreferred. If an aneuploid zygote is obtained, then the number ofchromosomes should not vary by more than one with respect to the euploidnumber of the organism from which either gamete originated.

[0146] In addition to similar biological considerations, physical onesalso govern the amount (e.g., volume) of exogenous genetic materialwhich can be added to the nucleus of the zygote or to the geneticmaterial which forms a part of the zygote nucleus. If no geneticmaterial is removed, then the amount of exogenous genetic material whichcan be added is limited by the amount which will be absorbed withoutbeing physically disruptive. Generally, the volume of exogenous geneticmaterial inserted will not exceed about 10 picoliters. The physicaleffects of addition must not be so great as to physically destroy theviability of the zygote. The biological limit of the number and varietyof DNA sequences will vary depending upon the particular zygote andfunctions of the exogenous genetic material and will be readily apparentto one skilled in the art, because the genetic material, including theexogenous genetic material, of the resulting zygote must be biologicallycapable of initiating and maintaining the differentiation anddevelopment of the zygote into a functional organism.

[0147] The number of copies of the transgene constructs which are addedto the zygote is dependent upon the total amount of exogenous geneticmaterial added and will be the amount which enables the genetictransformation to occur. Theoretically only one copy is required;however, generally, numerous copies are utilized, for example,1,000-20,000 copies of the transgene construct, in order to insure thatone copy is functional. As regards the present invention, there willoften be an advantage to having more than one functioning copy of eachof the inserted exogenous DNA sequences to enhance the phenotypicexpression of the exogenous DNA sequences.

[0148] Any technique which allows for the addition of the exogenousgenetic material into nucleic genetic material can be utilized so longas it is not destructive to the cell, nuclear membrane or other existingcellular or genetic structures. The exogenous genetic material ispreferentially inserted into the nucleic genetic material bymicroinjection. Microinjection of cells and cellular structures is knownand is used in the art.

[0149] Reimplantation is accomplished using standard methods. Usually,the surrogate host is anesthetized, and the embryos are inserted intothe oviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

[0150] Transgenic offspring of the surrogate host may be screened forthe presence and/or expression of the transgene by any suitable method.Screening is often accomplished by Southern blot or Northern blotanalysis, using a probe that is complementary to at least a portion ofthe transgene. Western blot analysis using an antibody against theprotein encoded by the transgene may be employed as an alternative oradditional method for screening for the presence of the transgeneproduct. Typically, DNA is prepared from tail tissue and analyzed bySouthern analysis or PCR for the transgene. Alternatively, the tissuesor cells believed to express the transgene at the highest levels aretested for the presence and expression of the transgene using Southernanalysis or PCR, although any tissues or cell types may be used for thisanalysis.

[0151] Alternative or additional methods for evaluating the presence ofthe transgene include, without limitation, suitable biochemical assayssuch as enzyme and/or immunological assays, histological stains forparticular marker or enzyme activities, flow cytometric analysis, andthe like. Analysis of the blood may also be useful to detect thepresence of the transgene product in the blood, as well as to evaluatethe effect of the transgene on the levels of various types of bloodcells and other blood constituents.

[0152] Progeny of the transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where matingwith a partner is to be performed, the partner may or may not betransgenic and/or a knockout; where it is transgenic, it may contain thesame or a different transgene, or both. Alternatively, the partner maybe a parental line. Where in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in vitro, orboth. Using either method, the progeny may be evaluated for the presenceof the transgene using methods described above, or other appropriatemethods.

[0153] The transgenic animals produced in accordance with the presentinvention will include exogenous genetic material. Further, in suchembodiments the sequence will be attached to a transcriptional controlelement, e.g., a promoter, which preferably allows the expression of thetransgene product in a specific type of cell.

[0154] Retroviral infection can also be used to introduce the transgeneinto a non-human animal. The developing non-human embryo can be culturedin vitro to the blastocyst stage. During this time, the blastomeres canbe targets for retroviral infection (Jaenich, R. (1976) PNAS73:1260-1264). Efficient infection of the blastomeres is obtained byenzymatic treatment to remove the zona pellucida (Manipulating the MouseEmbryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, 1986). The viral vector system used to introduce the transgeneis typically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

[0155] A third type of target cell for transgene introduction is theembryonal stem cell (ES). ES cells are obtained from pre-implantationembryos cultured in vitro and fused with embryos (Evans et al. (1981)Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler etal. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

[0156] The present invention is further illustrated by the followingexamples which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication) are hereby expressly incorporated by reference. Thepractice of the present invention will employ, unless otherwiseindicated, conventional techniques that are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, (2nd ed., Sambrook, Fritsch andManiatis, eds., Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,683,202;and Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds., 1984).

5. EXAMPLES Example 1 Genotyping

[0157] All human subjects were unrelated, Caucasian, healthy blooddonors from Sheffield (n=112). Subjects were typed at the loci indicatedin Table 1. TABLE 2 Markers Used in Haplotype Study Marker GeneReference 2221223 IL1A Todd& Naylor, Nucleic Acids Res. 19: 3756(1991)gz5/gz6 IL1A Zuliani, et al., Am. J. Hum. Genet. 46: 963-69 (1990)  −889 IL1A McDowell, et al., Arth. & Rheum. 38: 221-8(1995)  +3954 IL1Bdi Giovine, et al., Cytokine 7(6): 606 (1995)   −511 IL1B di Giovine,Hum. Molec. Genet. 1(6): 450 (1992) gaat.p33330 between IL1B and Murray,et al., Coop. Hum. Link. Center, unpublished IL1RN Y31 between IL1B andSpurr, et al., Cytogenet. & Cell Genet. 73: 255-73 (1996) 1L1RN VNTRIL1RN Tarlow, et al., Hum. Genet. 91: 403-4 (1993)

[0158] The primer sequences and fluorescent labels used in PCRamplification of markers were as in Table 3. TABLE 2 Primer Sequence andFlourescent Label for Genotyping Marker Label Primer Sequence 2221223HEX ATGTATAGAATTCCATTCCTG (SEQ ID NO. 8) TAAAATCAAGTGTTGATGTAG (SEQ IDNO. 9) gz51gz6 FAM GGGA7TACAGGCGTGAGCCACCGCG (SEQ ID NO. 10)TTAGTATTGCTGGTAGTATTCATAT (SEQ ID NO. 11) −889 NONETGTTCTACCACCTGAACTAGG (SEQ ID NO. 12) TTACATATGAGCCTTCCATG (SEQ ID NO.13) +3954 NONE CTCAGGTGTCCTCGAAGAAATCAAA (SEQ ID NO. 14)GCTTTMGCTGTGAGTCCCG (SEQ ID NO. 15) −511 NONE TGGCATTGATCTGGTTCATC (SEQID NO. 16) GTTTAGGAATCTTCCCACTT (SEQ ID NO. 17) gaat.p33330 FAMGAGGCGTGAGAATCTCAAGA (SEQ ID NO. 18) GTGTCCTCAAGTGGATCTGG (SEQ ID NO.19) Y31 HEX GGGCAACAGAGCAATGTTTCT (SEQ ID NO. 20) CAGTGTGTCAGTGTACTGTT(SEQ ID NO. 21) VNTR NONE CTCAGCAACACTCCTAT (SEQ ID NO. 22)TCCTGGTCTGCAGGTAA (SEQ ID NO. 23)

[0159] Reaction conditions were as described in Table 4. TABLE 4Reaction Conditions Marker Conditions 222/223 50 mM KCI, 10 mM Tris-HCIpH 9.0, 1.5 mM MgCI₂, 200: mM dNTPs, 25 ng primers, 50 ng template,0.004% W-1 (Gibco-BRL) 0.2 u Taq, PCR was done at 30 cycles of 94° C.for 1″. 55° C. for 1″, 72° C. for 1″ gz5/gz6 as per marker 2221223,except 1 u of Perfect Match (StrataGene) was added  −889 per marker222/223, except PCR was done for 1 cycle at 96° C. for 1″, 40 cycles of94° C. for 1″, 46° C. for 1″ 72° C. for 1″ and 1 cycle of 72° C. for 4″,products were cleaved with Ncol for analysis +3954 as per marker222/223, except PCR was done for 35 cycles with annealing at 67.5° C.,products were cleaved with Taq 1 for analysis  −511 as per marker2221223, except PCR was done for 1 cycle at 95° C. for 2″, 35 cycles of95° C. for 1″, 53° C. for 1″ 74° C. for 1″ and 1 cycle of 74° C. for 4″,products were cleaved with Aval and Bsu361 for analysis gaat.p33330 permarker 222/223 Y31 per marker 222/223 VNTR per marker 222/223 exceptwith 1.7 mM MgCI₂, 1 cycle at 96° C. for 1″; 30 cycles of 94° C. for 1″,60° C. for 1″, 70° C. for 1″ and 1 cycle at 70° C. for 2″

[0160] 2221223, gz5/gz6, gaat.p33330 and Y31 PCR products were examinedby agarose gel electrophoresis and the remainder of the PCR productswere pooled according to the intensity of ethidium bromide staining. 2gl of the pool was analyzed on an ABI 373A automated sequencer andallele sizes were determined using the Genescan and Genotyper software.Alleles were globally binned using a simple computer program andnumbered in order of size.

[0161] −889 PCR products were digested with NcoI and the resultingfragments sized on 8% PAGE. Allele 1 produces 83 and 16 bp fragments.Allele 2 produces a 99 bp fragment.

[0162] +3954 PCR products were digested with restriction enzyme Taq I.Allele 1 produces fragments of 97, 85 and 12 bp, and allele 2 producesfragments of 182 and 12 bp.

[0163] −511 PCR products were digested with AvaI and Bsu36I and thefragments were sized by 8% PAGE. Allele 1 produces 190 and 114 bpfragments when digested with AvaI and a 304 bp fragment when digestedwith Bsu36I. Allele 2 produces a 304 bp fragment when digested with AvaIand 190 and 114 bp fragments when digested with Bsu36I.

[0164] VNTR PCR products were sized by electrophoresis on 2% agarose gelat 90V for 45 minutes. Allele 1 has 4 repeats and the PCR product is 412bp, allele 2 has 2 repeats and the PCR product is 240 bp, allele 3 has 3repeats and the PCR product is 326 bp, allele 4 has 4 repeats and thePCR product is 498 bp, allele 5 has 6 repeats and the PCR product is 584bp.

[0165] Intergenic distances were determined by estimation based on theinsert sizes of relevant PAC clones from a contig spanning the IL-1 genecluster (Nicklin, et al., Genomics 19:382-4 (1994)). Intragenicdistances were determined from the relevant nucleotide sequence obtainedform the GENBANK database.

Example 2 Method for Estimating Linkage Disequilibrium

[0166] Because four of the markers studied herein are multiallelic, apreliminary analysis was carried out to determine which alleliccombinations between pairs of loci contributed to the greatestdisequilibrium, in order that the disequilibrium would not be maskedwhen the alleles were grouped into biallelic systems. The E. H. programof Xie and Ott (Handbook of Human Genetic Link-age, 1994, John HopkinsUniversity Press, 188-98), incorporated by reference herein, was used toestimate haplotype frequencies under H₀ (no linkage) and H₁ (alleliclinkage allowed). It was found that the elaborate allele grouping,strategy had some advantages over commonly used methods, in thatdisequilibrium was detected between almost all pairwise combinations ofmarkers examined and there was good correlation between disequilibriumand physical distance.

[0167] More specifically, the E.H. program of Xie and Ott was used todetermine maximum likelihood estimates of disequilibriumn (D_(j))between each pairwise combination of alleles, whereD_(ij)=h_(ij)−p_(i)q_(j) are the frequencies for allele i at locus 1 andallele j at locus 2 respectively, and h_(ij) is the frequency of thehaplotype ij. The program calculated maximum likelihood values for thehaplotype frequencies (and hence allele frequencies) under H₀ (noassociation) and haplotype frequencies under H₁ (allelic associationallowed). For markers with greater than two alleles, the E. H. estimatefor allele frequencies correlated poorly with the allele frequencies asestimated directly from the sample population, and therefore gave noconfidence to the D_(ij) estimates given. It was therefore necessary togroup alleles of the multi-allelic markers into a biallelic system.Analysis of the markers in a biallelic format has the added advantagesthat the notation {circumflex over ( )}D_(ij), p_(j), and q_(j) can besimplified to {circumflex over ( )}D, p, and q respectively, where p andq are defined to be the frequencies of the rarer alleles at both loci(such that without loss of generality p α q α 0.5), and {circumflex over( )}D is the estimated disequilibrium between those alleles.

[0168] Under a biallelic system, power is also much simpler to determineusing equations as detailed by Hill (Hill, Heredity, 33: 229-39 (1974)).In addition, the sign of {circumflex over ( )}D becomes informative,such that {circumflex over ( )}D>0 when the rarer alleles at each of thetwo loci are associated, and {circumflex over ( )}D<0 when the rareallele at one locus is associated with the common allele at the otherlocus.

[0169] Because the method of allele grouping clearly affected the powerto detect disequilibrium (Zouros, et al., Genet. 85: 543-50 (1977);Weir, et al., Genet. 88: 633-42 (1976)), a preliminary analysis wasconducted to ensure that the grouping did not mask disequilibriumbetween subsets of alleles. In this analysis,δ_(ij)=(O′_(ij)−E_(ij))//E_(ij) was calculated for each haplotype, whereE_(ij) is the expected number of haplotypes ij assuming equilibrium(E_(ij)=2n p_(i)q_(j), where n=number of individuals in the study), andO′_(ij) is a basic estimate for the observed haplotype count determinedas follows. All genotypes that could be unambiguously resolved werehaplotype counted. Each double heterozygote (i₁i₂/j₁j₂) could beresolved into two possible haplotype sets, [i₁J₁,i₂j₂] or [i₁J₂,i₂j₁].Using the haplotype frequencies as estimated from the unambiguoushaplotype count, the probability of each set was calculated and used asa “partial” count. In this way the ambiguous genotypes were alsohaplotype counted, and the total counts (ambiguous plus unambiguous)constituted the O′_(ij)'s used in δ_(ij). Once established, themagnitude and sign of the δ_(ij)'s were used to determine which alleliccombinations showed greatest deviation from the null hypothesis of noassociation. This information was used to group alleles at themultiallelic loci into biallelic systems to enable efficient use of theE.H. program.

[0170] In order to compare the degree of disequilibrium betweendifferent pairwise combinations of loci, a frequency independent measureof disequilibrium ˜D, the proportion of maximum possible disequilibriumin the given direction) was calculated, where ˜D={circumflex over( )}D/|D_(max)| (Thompson, et al., Am. J. Hum. Genet. 42: 113-24(1988)). The relationship between p and q are such that p α q α 0.5, andit can therefore be written that −pq α D α p(1−q) such that when{circumflex over ( )}D<0, D_(max)=−pq and when {circumflex over ( )}D>0,D_(max)=p(1−q). Output from the E.H. program included log-likelihoodsfor the maximum likelihood parameter values under H₀ and H₁, and since−21 n (L₀/L₁)˜X² ₁. where L₀ and L₁ are the likelihoods under H₀ and H₁,p- values could then be determined for each test.

[0171] The asymptotic variance for {circumflex over ( )}D, under H₀: D=0and H₁ were computed using the formula as defined by Hill (Heredity 33:229-39 (1974)) for genotypic data. Using these, the power for eachpairwise comparison could be calculated.

[0172] Common haplotypes containing all 8 loci were identified from thepreliminary analysis of δ_(ij) described above, and backed up by themagnitude and sign of the disequilibria once the alleles at themultiallelic loci had been grouped. For these loci, the allele in thegroup which contributed most to the disequilibrium has been identifiedon the haplotype. To estimate the population haplotype frequencies,rates of carriage of at least one copy of the relevant alleles in thepopulation were determined. These do not represent true haplotypes sincephase is unknown. Monte Carlo simulation techniques were used to testfor significant deviation from a simulated null distribution for thesecombined carriages under the assumption of no association.

Example 3 Estimation of Linkage Disequilibrium in the IL-1 Gene Cluster

[0173] A number of biallelic and multiallelic markers in and around theIL-1 genes have been identified. However, the extent of linkagedisequilibrium between the markers, and the prevalence of multimarkerhaplotypes in the general population have not until now been identified.

[0174]FIG. 1 shows the relative positions of the 8 marker loci used inthis study. DNA samples from 212 unrelated healthy volunteers weregenotyped for each of these markers, and the resulting estimates ofallele frequencies are shown in Table 5. TABLE 5 Estimated frequenciesof marker alleles 222/223 freq. gz5/gz6 freq. −889 freq. +3953 freq.1(126 bp) 0.005 1(79 bp) 0.003 1(Ncol) 0.714 1(2 Taql) 0.812 2 (128 bp)0.018 2 (82 bp) 0.005 2 0.286 2 0.188 3 (130 bp) 0.378 3 (88 bp) 0.676 4(132 bp) 0.299 4 (91 bp) 0.316 5 (134 bp) 0.016 6 (136 bp) 0.208 7 (138bp) 0.055 8 (140 bp) 0.003 9 (142 bp) 0.010 10 (144 bp) 0.008 *total 384392 398 398 −511 freq. gaat.p33330 freq. Y31 freq. VNTR freq. 1 0.618 1(189 bp) 0.658 1 (148 bp) 0.092 1 0.744 2 (Bsu361) 0.382 2 (193 bp)0.002 2 (158 bp) 0.008 2 0.256 3 (197 bp) 0.255 3 (160 bp) 0.454 4 (201bp) 0.084 4 (162 bp) 0.062 5 (164 bp) 0.003 6 (166 bp) 0.122 7 (168 bp)0.035 8 (170 bp) 0.030 9 (172 bp) 0.095 10 (174 bp) 0.087 11 (176 bp)0.003 12 (178 bp) 0.011 398 404 370 398

[0175] To determine the linkage disequilibria between pairwisecombinations of loci, the computer program of Xie and Ott was used. Thisprogram was found to be most efficient when used with biallelic systems,therefore alleles at the multiallelic loci were grouped in the most 5appropriate way for each pairwise comparison, such that disequilibriumbetween subsets of alleles was not masked.

[0176] In Table 6, the disequilibria between pairs of loci are expressedas ˜D, the ratio of {circumflex over ( )}D to its maximum value D_(max)and are shown together with the approximate physical distances betweenthe loci in kilobase pairs. TABLE 6 Disequilibrium (˜D = {circumflexover ( )}D/|D_(max)|) and physical distances between markers 222/223gz5/gz6 −889 +3953 222/223 — +0.872 +0.829 +0.710 gz5/gz6 2.5 — −0.889−0.695  −889 7 4.5 — +0.804 +3953 55 55 50 —  −511 60 60 55 4.5gaat.p33330 260 260 255 205 Y31 310 310 305 255 VNTR 380 380 375 325−511 gaat.p33330 Y31 VNTR 222/223 +0.535 +0433 +0.364 −0.499 gz5/gz6+0.540 +0.517 −0.503 +0.286  −889 −0.264 +0.337 +0.318 −0.207 +3954−0.617 +0.409 −0.475 0.439  −511 — +0.691 −0.456 +0.448 gaat.p33330 200— +0.639 +0.442 Y31 250 50 — −0.765 VNTR 320 120 70 —

[0177] Table 7 shows the power to detect 50% D_(max) for each locuscombination, and the p values for each corresponding D. TABLE 7 Power todetect 50% D_(max) and p values of −2Ln (L_(o)L₁) 222/223 gz5/gz6 −889+3953 22/223 — −100(+) −100(+) 98(+) gz5/gz6 <1 × 10⁻¹⁰ — 87(−) 60(−) −889 <1 × 10⁻¹⁰ <3 × 10⁻⁸ — −100(+) +3954 −1 × 10⁻⁷ *−9 × 10⁻³ <1 ×10⁻¹⁰ —  −511 −9 × 10⁻¹⁰ −4 × 10⁻¹⁰ *−9.4 × 10⁻² *−2.6 × 10⁻² gaat.p3330−9 × 10⁻⁸ −2 × 10⁻⁹ *−1.7 × 10⁻² −5 × 10⁻⁴ Y31 −1 × 10⁻⁴ −4 × 10⁻⁴ −6 ×10⁻⁴ −1 × 10⁻⁷ VNTR −1 × 10⁻³ −1 × 10⁻³ *−3 × 10⁻¹ *−1.2 × 10⁻¹ −511gaat.p33330 Y31 VNTR 22/223 ˜100(+) ˜100(+) ˜100(+) 93(−) gz5/gz6˜100(+) ˜100(+) 98(−) ˜100(+)  −889 96(−) 89(+) ˜100(+) 78(−) +395479(−) 97(+) ˜100(+) 52(−)  −511 — ˜100(+) ˜100(−) ˜100(+) gaat.p33330 <1× 10⁻¹⁰ — 49(+) ˜100(+) Y31 ˜2 × 10⁻⁴ *˜7 × 10⁻³ — 89(−) VNTR ˜8 × 10⁻⁶˜1 × 10⁻⁹ 2 × 10⁻⁷ —

[0178] Significant linkage disequilibrium (p_(corr)<0.05) was detectedbetween most combinations of loci, with only a few exceptions. Theseinclude the comparisons between the VNTR and the more distant biallelicmarkers, +3954, and −889, in which the disequilibrium is in the negativedirection and consequently the power is reduced (Table 7). Thecorrelation between disequilibrium ˜D and physical distance was r=−0.752(p<0.0001, one tailed) (FIG. 2).

[0179] In order to compare different grouping methods for themultiallelic markers, ˜D was calculated for all the comparisonsinvolving 222/223 using two additional grouping strategies. The first ofthese was a “common allele versus the rest” approach, and the second wasa grouping based on allele size, using the bimodal distribution ofallele frequency versus size which was observed for all the multiallelicmarkers examined. The results of this analysis are shown in Table 8,where ˜D values for the three grouping methods are compared. TABLE 8 ˜Dvalues for three methods of grouping alleles at the multiallelic markerloci δ_(ij) common vs. rest allele size gz5/gz6 0.87 0.79 0.77  −8990.83 0.81 0.98 +3954 0.71 *0.74 0.77  −511 0.54 *0.15 0.61 gaat.p333300.43 *0.03 0.53 Y31 0.36 *0.12 0.16 VNTR 0.5 0.48 *0.04

[0180] It can be seen that the disequilibrium is not detected in severalinstances using these other grouping strategies, notably 222/223 with−511 and gaat.p33330 in the common versus rest approach, 222/223 withY31 in both the common versus rest and allele size approaches, and222/223 with VNTR in the allele size approach.

[0181] Examination of which alleles of the multiallelic loci werecontributing greatest to the disequilibrium, from the determination ofδ_(ij) revealed the existence of 2 haplotypes containing alleles of all8 loci. These were confirmed by examination of the haplotype frequenciesand disequilibrium values obtained after the grouping. The firsthaplotype: alleles 44112332 (expressed in chromosome order, see FIG. 1)is the most common (carriage of 34/198), and is present 7 times morefrequently than expected (expected=4.5/198) (p<0.000001). The secondhaplotype: alleles 33221461 (carriage of 2/206) was present 4 times morefrequently than expected (expected 0.5/206), but this was notstatistically significant (p˜0.106). However, examination of a largersample size might assist in increasing the statistical significance ofthis finding.

[0182] The data presented indicate a significant degree of linkagedisequilibrium across an approximately 400 Kb stretch of chromosome2q13. The disequilibrium was strong both for the three markers withinthe IL-1α gene, as might be expected, but was also strong between someof the more distantly separated markers (−899/+3954;˜D=+0.804, physicaldistance=50 Kb) (Table 6). However, ˜D was considerably diminishedbetween the extreme ends of the cluster. Within the IL-1β gene, amoderate value of ˜D (+3954/−511; ˜D=−0.617) was obtained, although thiswas not significant when corrected for multiple comparisons, probablyreflecting the reduction in power when disequilibrium is in the negativedirection (Thompson, et al, Am J Hum. Genet. 42: 113-24 (1988)).

[0183] Overall, there is a good correlation between physical distanceand linkage disequilibrium (FIG. 2); r=−0.752. The reliability of ritself depends partly on the reliability of the estimates of bothphysical distance and ˜D. Over the short distances, the physicaldistances are accurate since they are determined from known DNAsequence, whereas the longer range estimates are less precise. The powercan be taken tentatively as one indicator of the reliability of ˜D,since if power is low this indicates that the sample size was too small,and with low sample sizes the estimates for ˜D may be unreliable.

[0184] The success of the elaborate grouping strategy is indicated byTable 8, which shows several instances where disequilibrium betweenparticular loci is apparently low or not detected when other commonlyused grouping methods are employed. The disadvantages of the groupingstrategy used here are that it is rather laborious since the informationused for the grouping was based on an approximate estimate of the“observed” haplotype frequencies (see Example 2). For the morepolymorphic markers the higher heterozygosity meant that the estimate ofδ_(ij), was less precise since there was a higher proportion ofambiguous haplotypes. Notwithstanding this drawback, care was taken totake into account both the sign and magnitude of δ_(ij), and thefrequencies of the alleles concerned.

[0185] The method could be simplified, in a sufficiently large study, byjust considering the unambiguous haplotypes when determining thegrouping. The determination of δ_(ij) uses the maximum amount of priorknowledge for the grouping of the multiallelic markers, and this may bethe reason why disequilibrium between almost all pairwise combinationsof markers was detected.

[0186] The two haplotypes containing all 8 markers, as well as othershorter haplotypes, are of particular interest since it is likely thatparticular combinations of alleles of the IL-I genes may act in concertto determine an overall inflammatory phenotype. An understanding ofwhich markers are in strong linkage disequilibrium not only allows formore rational design of genetic studies but also may provide clues todisease mechanism. Therefore, in addition to the alleles identifiedherein, the IL-1 (44112332) haplotype may contain the following alleles:

[0187] allele 2 of the 1731 marker of the IL1RN gene(A at position1731);

[0188] allele 2 of the 1812 marker of the IL1RN gene (A at position1812);

[0189] allele 2 of the 1868 marker of the IL1RN gene(G at position1868);

[0190] allele 2 of the 1887 marker of the IL1RN gene(C at position1887);

[0191] allele 2 of the 8006 marker of the IL1RN gene (contains a HpaIIor MspI site)

[0192] allele 2 of the 8061 marker of the IL1RN gene (lacks a MwoI site)

[0193] allele 2 of the 9589 marker of the IL1RN gene (contains an SspIsite)

[0194] Furthermore, the following PCR primers may be used to amplifythese alleles: TTACGCAGATAAGAACCAGTTTGG (SEQ ID NO. 24)TTTCCTGGACGCTTGCTCACCA (SEQ ID NO. 25) (used for 1731, 1812, 1868, and1887) TTCTATCTGAGGAACAACCAACTAGTAGC (SEQ ID NO. 26)CACCAGACTTGACACAGGACAGGCACATC (SEQ ID NO. 27) (used for 8006)CGACCCTCTGGGAGAAAATCCAGCAAG (SEQ ID NO. 28) (used with SEQ ID NO. 20 for8006) ACACAGGAAGGTGCCAAGCA (SEQ ID NO. 29) TGCAGACAGACGGGCAAAGT (SEQ IDNO. 30) (used for 8006 and 9589) TTGTGGGGACCAGGGGAGAT, (SEQ ID NO. 31)and AGCCTGGCACTCTGCTGAAT (SEQ ID NO. 32) (used for 9589).

Example 4 The IL-1 (441123-32) Haplotype is Associated with DiabeticNephropathy

[0195] The presence of the two haplotypes described herein wasinvestigated in healthy and diseased populations to determine if thehaplotypes were associated with inflammatory disease. 81 non-insulindependant diabetes mellitus (NIDDM) patients with nephropathy werecompared with 198 ethnically matched healthy subjects in example 3 and147 NIDDM patients without nephropathy. Genotyping was carried out as inexample 1.

[0196] The IL-1 (44112332) haplotype was carried by 24 of 79 of theNIDDM nephropathy patients and 25 of 141 NIDDM without nephropathypatients. However, the second haplotype (33221461) was not found in thenephropathy patients (0/8 1). The IL-1 (44112332) haplotype wassignificantly over represented in the patient group compared with thehealthy control group (24/79 vs. 34/198; p=0.015) and the NIDDM withoutnephropathy group (24/79 vs. 25/141; p=0.03).

Example 5 An IL-1 Haplotype is Associated with Inflammatory Disease

[0197] This is a prophetic example. Other diseases are examined as perExample 4. The IL-1 (44112332) haplotype is found to be associated withcoronary artery disease, osteoporosis, nephropathy in diabetes mellitus,alopecia areata, Graves disease, systemic lupus erythematosus, lichensclerosis and ulcerative colitis.

[0198] Likewise, the IL-1 (33221461) haplotype is associated withperiodontal disease, juvenile chronic arthritis, psoriasis, insulindependant diabetes and diabetic retinopathy.

Example 6 Novel Markers are Linked to an IL-1 Haplotype

[0199] This is a prophetic example. Additional markers are identified bysequence and restriction enzyme analysis of the 2q13-14 region. Thesenew markers are identified as belonging to an IL-1 haplotype in themanner described in Examples 2 and 3.

Example 7 The IL-1 (44112332) Haplotype Is Used to Predict DiseaseSusceptibility

[0200] This is a prophetic example. A patient with a family history ofulcerative colitis is genotyped for the presence of the IL-1 (44112332)haplotype. Genotyping is performed as in Example I and the patient isdetermined to carry one or more alleles of the haplotype. The patient isthus treated with IL-1 antagonists to prevent disease.

[0201] A second patient with a family history of coronary artery diseaseis genotyped at the IL-1 gene cluster. The patient is found to carry oneor more alleles; of the IL-1 (44112332) haplotype and be homozygous forthe VNTR allele 2. Thus, the patient is 5.4 times as likely to developcoronary artery disease as the general population and is treatedvigorously to prevent disease.

Example 8 Additional Haplotypes are Statistically Significant

[0202] This is a prophetic example. An additional 400 chromosomes aretyped as per Example 1 and linkage disequilibrium assessed as perExample 2. The IL-1 (33221461) haplotype is found to be present about 4times more frequently than expected (p−0.05).

[0203] In a similar manner, the following markers are determined to bepresent in the IL-1 (44112332) haplotype (p<<0.05).

[0204] allele 2 of the 1731 marker of the IL1RN gene

[0205] allele 2 of the 1812 marker of the IL1RN gene

[0206] allele 2 of the 1868 marker of the IL1RN gene

[0207] allele 2 of the 1887 marker of the IL1RN gene

[0208] allele 2 of the 8006 marker of the IL1RN gene

[0209] allele 2 of the 8061 marker of the IL1RN gene

[0210] allele 2 of the 9589 marker of the IL1RN gene

1 32 1 11970 DNA Homo sapiens 1 aagcttctac cctagtctgg tgctacacttacattgctta catccaagtg tggttatttc 60 tgtggctcct gttataacta ttatagcaccaggtctatga ccaggagaat tagactggca 120 ttaaatcaga ataagagatt ttgcacctgcaatagacctt atgacaccta accaacccca 180 ttatttacaa ttaaacagga acagagggaatactttatcc aactcacaca agctgttttc 240 ctcccagatc catgcttttt tgcgtttattattttttaga gatgggggct tcactatgtt 300 gcccacactg gactaaaact ctgggcctcaagtgattgtc ctgcctcagc ctcctgaata 360 gctgggacta caggggcatg ccatcacacctagttcattt cctctattta aaatatacat 420 ggcttaaact ccaactggga acccaaaacattcatttgct aagagtctgg tgttctacca 480 cctgaactag gctggccaca ggaattataaaagctgagaa attctttaat aatagtaacc 540 aggcaacatc attgaaggct catatgtaaaaatccatgcc ttcctttctc ccaatctcca 600 ttcccaaact tagccactgg ttctggctgaggccttacgc atacctcccg gggcttgcac 660 acaccttctt ctacagaaga cacaccttgggcatatccta cagaagacca ggcttctctc 720 tggtccttgg tagagggcta ctttactgtaacagggccag ggtggagagt tctctcctga 780 agctccatcc cctctatagg aaatgtgttgacaatattca gaagagtaag aggatcaaga 840 cttctttgtg ctcaaatacc actgttctcttctctaccct gccctaacca ggagcttgtc 900 accccaaact ctgaggtgat ttatgccttaatcaagcaaa cttccctctt cagaaaagat 960 ggctcatttt ccctcaaaag ttgccaggagctgccaagta ttctgccaat tcaccctgga 1020 gcacaatcaa caaattcagc cagaacacaactacagctac tattagaact attattatta 1080 ataaattcct ctccaaatct agccccttgacttcggattt cacgatttct cccttcctcc 1140 tagaaacttg ataagtttcc cgcgcttccctttttctaag actacatgtt tgtcatctta 1200 taaagcaaag gggtgaataa atgaaccaaatcaataactt ctggaatatc tgcaaacaac 1260 aataatatca gctatgccat ctttcactattttagccagt atcgagttga atgaacatag 1320 aaaaatacaa aactgaattc ttccctgtaaattccccgtt ttgacgacgc acttgtagcc 1380 acgtagccac gcctacttaa gacaattacaaaaggcgaag aagactgact caggcttaag 1440 ctgccagcca gagagggagt catttcattggcgtttgagt cagcaaaggt attgtcctca 1500 catctctggc tattaaagta ttttctgttgttgtttttct ctttggctgt tttctctcac 1560 attgccttct ctaaagctac agtctctcctttcttttctt gtccctccct ggtttggtat 1620 gtgacctaga attacagtca gatttcagaaaatgattctc tcattttgct gataaggact 1680 gattcgtttt actgagggac ggcagaactagtttcctatg agggcatggg tgaatacaac 1740 tgaggcttct catgggaggg aatctctactatccaaaatt attaggagaa aattgaaaat 1800 ttccaactct gtctctctct tacctctgtgtaaggcaaat accttattct tgtggtgttt 1860 ttgtaacctc ttcaaacttt cattgattgaatgcctgttc tggcaataca ttaggttggg 1920 cacataagga ataccaacat aaataaaacattctaaaaga agtttacgat ctaataaagg 1980 agacaggtac atagcaaact aattcaaaggagctagaaga tggagaaaat gctgaatgtg 2040 gactaagtca ttcaacaaag ttttcaggaagcacaaagag gaggggctcc cctcacagat 2100 atctggatta gaggctggct gagctgatggtggctggtgt tctctgttgc agaagtcaag 2160 atggccaaag ttccagacat gtttgaagacctgaagaact gttacaggta aggaataaga 2220 tttatctctt gtgatttaat gagggtttcaaggctcacca gaatccagct aggcataaca 2280 gtggccagca tgggggcagg ccggcagaggttgtagagat gtgtactagt cctgaagtca 2340 gagcaggttc agagaagacc cagaaaaactaagcattcag catgttaaac tgagattaca 2400 ttggcaggga gaccgccatt ttagaaaaattatttttgag gtctgctgag ccctacatga 2460 atatcagcat caacttagac acagcctctgttgagatcac atgccctgat ataagaatgg 2520 gttttactgg tccattctca ggaaaacttgatctcattca ggaacaggaa atggctccac 2580 agcaagctgg gcatgtgaac tcacatatgcaggcaaatct cactcagatg tagaagaaag 2640 gtaaatgaac acaaagataa aattacggaacatattaaac taacatgatg tttccattat 2700 ctgtagtaaa tactaacaca aactaggctgtcaaaatttt gcctggatat tttactaagt 2760 ataaattatg aaatctgttt tagtgaatacatgaaagtaa tgtgtaacat ataatctatt 2820 tggttaaaat aaaaaggaag tgcttcaaaacctttctttt ctctaaagga gcttaacatt 2880 cttccctgaa cttcaattaa agctcttcaatttgttagcc aagtccaatt tttacagata 2940 aagcacaggt aaagctcaaa gcctgtcttgatgactacta attccagatt agtaagatat 3000 gaattactct acctatgtgt atgtgtagaagtccttaaat ttcaaagatg acagtaatgg 3060 ccatgtgtat gtgtgtgacc cacaactatcatggtcatta aagtacattg gccagagacc 3120 acatgaaata acaacaatta cattctcatcatcttatttt gacagtgaaa atgaagaaga 3180 cagttcctcc attgatcatc tgtctctgaatcaggtaagc aaatgactgt aattctcatg 3240 ggactgctat tcttacacag tggtttcttcatccaaagag aacagcaatg acttgaatct 3300 taaatacttt tgttttaccc tcactagagatccagagacc tgtctttcat tataagtgag 3360 accagctgcc tctctaaact aatagttgatgtgcattggc ttctcccaga acagagcaga 3420 actatcccaa atccctgaga actggagtctcctggggcag gcttcatcag gatgttagtt 3480 atgccatcct gagaaagccc cgcaggccgcttcaccaggt gtctgtctcc taacgtgatg 3540 tgttgtggtt gtcttctctg acaccagcatcagaggttag agaaagtctc caaacatgaa 3600 gctgagagag aggaagcaag ccagctgaaagtgagaagtc tacagccact catcaatctg 3660 tgttattgtg tttggagacc acaaatagacactataagta ctgcctagta tgtcttcagt 3720 actggcttta aaagctgtcc ccaaaggagtatttctaaaa tattttgagc attgttaagc 3780 agatttttaa cctcctgaga gggaactaattggaaagcta ccactcacta caatcattgt 3840 taacctattt agttacaaca tctcatttttgagcatgcaa ataaatgaaa aagtcttcct 3900 aaaaaaatca tctttttatc ctggaaggaggaaggaaggt gagacaaaag ggagagaggg 3960 agggaagcct aatgaaacac cagttacctaagaccagaat ggagatcctc ctcactacct 4020 ctgttgaata cagcacctac tgaaagaactttcattccct gaccatgaac agcctctcag 4080 cttctgtttt ccttcctcac agaaatccttctatcatgta agctatggcc cactccatga 4140 aggctgcatg gatcaatctg tgtctctgagtatctctgaa acctctaaaa catccaagct 4200 taccttcaag gagagcatgg tggtagtagcaaccaacggg aaggttctga agaagagacg 4260 gttgagttta agccaatcca tcactgatgatgacctggag gccatcgcca atgactcaga 4320 ggaaggtaag gggtcaagca caataatatctttcttttac agttttaagc aagtagggac 4380 agtagaattt aggggaaaat taaacgtggagtcagaataa caagaagaca accaagcatt 4440 agtctggtaa ctatacagag gaaaattaatttttatcctt ctccaggagg gagaaatgag 4500 cagtggcctg aatcgagaat acttgctcacagccattatt tcttagccat attgtaaagg 4560 tcgtgtgact tttagccttt caggagaaagcagtaataag accacttacg agctatgttc 4620 ctctcatact aactatgcct ccttggtcatgttacataat cttttcgtga ttcagtttcc 4680 tctactgtaa aatggagata atcagaatcccccactcatt ggattgttgt aaagattaag 4740 agtctcaggc tttacagact gagctagctgggccctcctg actgttataa agattaaatg 4800 agtcaacatc ccctaacttc tggactagaataatgtctgg tacaaagtaa gcacccaata 4860 aatgttagct attactatca ttattattattattttattt tttttttttg agatggagtc 4920 tggctctgtc acccaggctg gagtgcagtggcacaatctc ggctcactgc aagctctgcc 4980 tcctgggttc atgccattct cctgcctcagcctcccgagt aagctgggaa tacaggcacc 5040 cgccactgtt cccggctaat tttttgtatttttagtagag acggagtttc accgtggtct 5100 ccatctcctc gtgatccacc caccttggcctcccaaagtg ccgggattac aggcgtgagc 5160 caccgcgccc ggcctattat tattattattactactacta ctacctatat gaatactacc 5220 agcaatacta atttattaat gactggattatgtctaaacc tcacaagaat cctaccttct 5280 cattttacat aaaaggaaac taagctcattgagataggta aactgcccaa tggcatacat 5340 ctgtaagtgg gagagcctca aatctaattcagttctacct gagtaaaaaa atcatggttt 5400 ctcctccatc cctttactgt acaagcctccacatgaacta taaacccaat attcctgttt 5460 ttaagataat acctaagcaa taacgcatgttcacctagaa ggttttaaaa tgtaacaaaa 5520 tataagaaaa taaaaatcac tcatatcgtcagtgagagtt tactactgcc agcactatgg 5580 tatgtttcct taaaatcttt gctatacacatacctacatg tgaacaaata tgtctaacat 5640 caagaccaca ctatttacaa ctttatatccagcttttctt acttagcaat gtattgagga 5700 cattttagag tgcccgtttt tcaccattataagcaatgca acaatgaaca tctgtataaa 5760 taaatattca tttctctcac cctttatttccttagaatat attcctagaa gtagaatttc 5820 ccagagccat gaggatttgt gacgctattgatatgtgcca ctttgcactc tctgtgacat 5880 atataattat ttttaatgca ttcatttttttctcagagtg cattcgtttg aaaacataga 5940 cgggaaatac tggtagtctt ccttgtcagttagaaacacc caaacaatga aaaatgaaaa 6000 agttgcacaa atagtctcta aaaacaatgaaactattgcc tgaggaattg aagtttaaaa 6060 agaagcacat aagcaacaac aaggataatcctagaaaacc agttctgctg actgggtgat 6120 ttcacttctc tttgcttcct catctggattggaatattcc taataccccc tccagaacta 6180 ttttccctgt ttgtactaga ctgtgtatatcatctgtgtt tgtacataga cattaatctg 6240 cacttgtgat catggtttta gaaatcatcaagcctaggtc atcacctttt agcttcctga 6300 gcaatgtgaa atacaacttt atgaggatcatcaaatacga attcatcctg aatgacgccc 6360 tcaatcaaag tataattcga gccaatgatcagtacctcac ggctgctgca ttacataatc 6420 tggatgaagc aggtacatta aaatggcaccagacatttct gtcatcctcc cctcctttca 6480 tttacttatt tatttatttc aatctttctgcttgcaaaaa acatacctct tcagagttct 6540 gggttgcaca attcttccag aatagcttgaagcacagcac ccccataaaa atcccaagcc 6600 agggcagaag gttcaactaa atctggaagttccacaagag agaagtttcc tatctttgag 6660 agtaaagggt tgtgcacaaa gctagctgatgtactacctc tttggttctt tcagacattc 6720 ttaccctcaa ttttaaaact gaggaaactgtcagacatat taaatgattt actcagattt 6780 acccagaagc caatgaagaa caatcactctcctttaaaaa gtctgttgat caaactcaca 6840 agtaacacca aaccaggaag atctttattatctctgataa catatttgtg aggcaaaacc 6900 tccaataagc tacaaatatg gcttaaaggatgaagtttag tgtccaaaaa cttttatcac 6960 acacatccaa ttttcatggc ggacatgttttagtttcaac agtatacata ttttcaaagg 7020 tccagagagg caattttgca ataaacaagcaagacttttt ctgattggat gcacttcagc 7080 taacatgctt tcaactctac atttacaaattattttgtgt tctatttttc tacttaatat 7140 tatttctgca attttcccaa tattgacatcgtgtatgtat ttgccatttt taatatcact 7200 agacaattca atcaggttgc tacgttggtcccttgggttt actctaaata gcttgattgc 7260 aaatatcttt gtatatatta ttgttttttctcctatcttg taatttcttt gagcacatcc 7320 caaagaggaa tgcctagatc aatgggcacaaataatttga cagctcttat taaacattat 7380 tctgtaagta aaaactgaac tacttttcagtatcactagc aacatatgag tgtatcagct 7440 tcctaaaccc ctccatgtta ggtcattatgaacttatgat ctaacaaatt acagggtctt 7500 atcccactaa tgaaattata agagattcaacacttattca gccccgaagg attcattcaa 7560 cgtagaaaat tctaagaaca ttaaccaagtatttacctgc ctagtgagtg tggaagacat 7620 tgtgaaggac acaaagatgt atagaattccattcctgact tccaggtatt tacaccatag 7680 gtggggacct aactacacac acacacacacacacacacac acacacacac accatgcaca 7740 cacaatctac atcaacactt gattttatacaaatacaatg aatttacttt ctttttggtt 7800 cttctcttca ccagtgaaat ttgacatgggtgcttataag tcatcaaagg atgatgctaa 7860 aattaccgtg attctaagaa tctcaaaaactcaattgtat gtgactgccc aagatgaaga 7920 ccaaccagtg ctgctgaagg tcagttgtcctttgtctcca acttaccttc atttacatct 7980 catatgtttg taaataagcc caataggcagacacctctaa caaggtgaca ctgtcctctt 8040 tccttcctac cacagccccc acctacccaccccactccca ttgattccag aggcgtgcct 8100 aggcaggatc tatgagaaaa tataacagagagtaagagga aaattacctt ctttcttttt 8160 cctttccctg cctgacctta ttcacctcccatcccagagc atccatttat tccattgatc 8220 tttactgaca tctattatct gacctacacaatactagaca ttaggacaat gtggcctgcc 8280 tccaagaaac tcaaataagc caactgagatcagagaggat taatcacctg ccaatgggca 8340 caaagcaaca agctgggagc caagtcccaaaatggggcct gctgcttcca gttcccctct 8400 ctctgcattg atgtcagcat tatccttcgtcccagtcctg tctccactac cactttcccc 8460 ctcaaacaca cacacacaca acagccttagatgttttctc cactgataag taggtgactc 8520 aatttgtaag tatataatcc aagaccttctattcccaagt agaatttatg tgcctgcctg 8580 tgcttttcta cctggatcaa gtgatgtctacagagtaggg cagtagcttc attcatgaac 8640 tcattcaaca agcattattc actgagagccttgtattttt caggcatagt gccaacagca 8700 gtgtggacag tggtgcatca aagcctctagtctcatagaa cttagtcttc tggaggatat 8760 ggaaaacaga caacccaaac aaccaacaaaagagcaagat gctgcaaaaa aaaaaaaaat 8820 gaatagggtg ctaagataga gaaaagtgggagagtgctat ttagacaaag tggtaaaaac 8880 aaagcccctt gtgagatgag agctgccgacagagggggcg ggtcatggtt gtgggttttt 8940 gggtaggaca ttcagaggag ggggcgggtcgtggttgtgg gtttttgggt aggacattca 9000 gaggaggggg cgggtcgtgg ttgtgggtttttgggtagga cattcagagg agggggcggg 9060 tcgtggttgt gggtttttgg gtaggacattcagaggaggg ggcgggtcgt ggttgtgggt 9120 ttttgggaca ttcagaggag tctgaatgcacccaggccta caacttcaag atggtaaagg 9180 acagctccaa ggatcagaag aagcattcttggaactgggg cattttgaga aggaggaaaa 9240 atatgcagag actagtgctt gcagagcttgcatttggatt tcatttgagg tacaatgaaa 9300 acccattaat gggtttcaca cagtgcaatggcctgacctc acttatattt cctaaaatag 9360 aaaacagatc agaaggaagg caatagagaagcagaaagtc caatgaggag gtttcacagc 9420 agtcatgggg gtggggtaag gaaaagaagtggaaagaaac agacagaatt gggttatatt 9480 ttggagatag aaccaacaga aggaagaggagaaacaacat ttactgagaa gggaaaaagt 9540 aggagaggaa taggtttggg aaataaatcctgctgacatt ggaaacccca aggaagcctc 9600 aaaagtatat ttacttgctt tagatttaaaagaataggaa agaagcatct caacttggaa 9660 tttgaaatct atttttccat aaaagtattgttaaattcta ctcatactca caagaaaagt 9720 acattctaaa gagtatattg aaagagtttactgatatact taggaatttt gtgtgtatgt 9780 gtgtgtgtgt atgtgtgtgt gtgtgtttaaccttcaattg ttgacttaaa tactgagata 9840 aatgtcatct aaatgctaaa ttgatttcccaaaggtatga tttgttcact tggagatcaa 9900 aatgtttagg gggcttagaa tcactgtagtgctcagattt gatgcaaaat gtcttaggcc 9960 tatgttgaag gcaggacaga aacaatgtttccctcctacc tgcctggata cagtaagata 10020 ctagtgtcac tgacaatctt cataactaatttagatctct ctccaatcaa ctaaggaaat 10080 caactcttat taatagactg ggccacacatctactaggca tgtaataaat gcttgctgaa 10140 tgaacaaatg aatgaagagc ctatagcatcatgttacagc catagtccta aagtggtgtt 10200 tctcatgaag gccaaatgct aagggattgagcttcagtcc tttttctaac atcttgttct 10260 ctaacagaat tctcttcttt tcttcataggagatgcctga gatacccaaa accatcacag 10320 gtagtgagac caacctcctc ttcttctgggaaactcacgg cactaagaac tatttcacat 10380 cagttgccca tccaaacttg tttattgccacaaagcaaga ctactgggtg tgcttggcag 10440 gggggccacc ctctatcact gactttcagatactggaaaa ccaggcgtag gtctggagtc 10500 tcacttgtct cacttgtgca gtgttgacagttcatatgta ccatgtacat gaagaagcta 10560 aatcctttac tgttagtcat ttgctgagcatgtactgagc cttgtaattc taaatgaatg 10620 tttacactct ttgtaagagt ggaaccaacactaacatata atgttgttat ttaaagaaca 10680 ccctatattt tgcatagtac caatcattttaattattatt cttcataaca attttaggag 10740 gaccagagct actgactatg gctaccaaaaagactctacc catattacag atgggcaaat 10800 taaggcataa gaaaactaag aaatatgcacaatagcagtt gaaacaagaa gccacagacc 10860 taggatttca tgatttcatt tcaactgtttgccttctgct tttaagttgc tgatgaactc 10920 ttaatcaaat agcataagtt tctgggacctcagttttatc attttcaaaa tggagggaat 10980 aatacctaag ccttcctgcc gcaacagttttttatgctaa tcagggaggt cattttggta 11040 aaatacttct cgaagccgag cctcaagatgaaggcaaagc acgaaatgtt attttttaat 11100 tattatttat atatgtattt ataaatatatttaagataat tataatatac tatatttatg 11160 ggaacccctt catcctctga gtgtgaccaggcatcctcca caatagcaga cagtgttttc 11220 tgggataagt aagtttgatt tcattaatacagggcatttt ggtccaagtt gtgcttatcc 11280 catagccagg aaactctgca ttctagtacttgggagacct gtaatcatat aataaatgta 11340 cattaattac cttgagccag taattggtccgatctttgac tcttttgcca ttaaacttac 11400 ctgggcattc ttgtttcatt caattccacctgcaatcaag tcctacaagc taaaattaga 11460 tgaactcaac tttgacaacc atgagaccactgttatcaaa actttctttt ctggaatgta 11520 atcaatgttt cttctaggtt ctaaaaattgtgatcagacc ataatgttac attattatca 11580 acaatagtga ttgatagagt gttatcagtcataactaaat aaagcttgca acaaaattct 11640 ctgacacata gttattcatt gccttaatcattattttact gcatggtaat tagggacaaa 11700 tggtaaatgt ttacataaat aattgtatttagtgttactt tataaaatca aaccaagatt 11760 ttatattttt ttctcctctt tgttagctgccagtatgcat aaatggcatt aagaatgata 11820 atatttccgg gttcacttaa agctcatattacacatacac aaaacatgtg ttcccatctt 11880 tatacaaact cacacataca gagctacattaaaaacaact aataggccag gcacggtggc 11940 tcagacctgt aatcccagca ctttgggagg11970 2 9721 DNA Homo sapiens “n” bases throughout the sequence may beA, T, C, G, other or unknown 2 agaaagaaag agagagagaa agaaaagaaagaggaaggaa ggaaggaagg aagaaagaca 60 ggctctgagg aaggtggcag ttcctacaacgggagaacca gtggttaatt tgcaaagtgg 120 atcctgtgga ggcanncaga ggagtcccctaggccaccca gacagggctt ttagctatct 180 gcaggccaga caccaaattt caggagggctcagtgttagg aatggattat ggcttatcaa 240 attcacagga aactaacatg ttgaacagcttttagatttc ctgtggaaaa tataacttac 300 taaagatgga gttcttgtga ctgactcctgatatcaagat actgggagcc aaattaaaaa 360 tcagaaggct gcttggagag caagtccatgaaatgctctt tttcccacag tagaacctat 420 ttccctcgtg tctcaaatac ttgcacagaggctcactccc ttggataatg cagagcgagc 480 acgatacctg gcacatacta atttgaataaaatgctgtca aattcccatt cacccattca 540 agcagcaaac tctatctcac ctgaatgtacatgccaggca ctgtgctaga cttggctcaa 600 aaagatttca gtttcctgga ggaaccaggagggcaaggtt tcaactcagt gctataagaa 660 gtgttacagg ctggacacgg tggctcacgcctgtaatccc aacatttggg aggccgaggc 720 gggcagatca caaggtcagg agatcgagaccatcctggct aacatggtga aaccctgtct 780 ctactaaaaa tacaaaaaat tagccgggcgttggcggcag gtgcctgtag tcccagctgc 840 tggggaggct gaggcaggag aatggtgtgaacccgggagg cggaacttgc agggggccga 900 gatcgtgcca ctgcactcca gcctgggcgacagagtgaga ctctgtctca aaaaaaaaaa 960 aaaagtgtta tgatgcagac ctgtcaaagaggcaaaggag ggtgttccta cactccaggc 1020 actgttcata acctggactc tcattcattctacaaatgga gggctcccct gggcagatcc 1080 ctggagcagg cactttgctg gtgtctcggttaaagagaaa ctgataactc ttggtattac 1140 caagagatag agtctcagat ggatattcttacagaaacaa tattcccact tttcagagtt 1200 caccaaaaaa tcattttagg cagagctcatctggcattga tctggttcat ccatgagatt 1260 ggctagggta acagcacctg gtcttgcagggttgtgtgag cttatctcca gggttgcccc 1320 aactccgtca ggagcctgaa ccctgcataccgtatgttct ctgccccagc caagaaaggt 1380 caattttctc ctcagaggct cctgcaattgacagagagct cccgaggcag agaacagcac 1440 ccaaggtaga gacccacacc ctcaatacagacagggaggg ctattggccc ttcattgtac 1500 ccatttatcc atctgtaagt gggaagattcctaaacttaa gtacaaagaa gtgaatgaag 1560 aaaagtatgt gcatgtataa atctgtgtgtcttccacttt gtcccacata tactaaattt 1620 aaacattctt ctaacgtggg aaaatccagtattttaatgt ggacatcaac tgcacaacga 1680 ttgtcaggaa aacaatgcat atttgcatggtgatacattt gcaaaatgtg tcatagtttg 1740 ctactccttg cccttccatg aaccagagaattatctcagt ttattagtcc cctcccctaa 1800 gaagcttcca ccaatactct tttcccctttcctttaactt gattgtgaaa tcaggtattc 1860 aacagagaaa tttctcagcc tcctacttctgcttttgaaa gctataaaaa cagcgaggga 1920 gaaactggca gataccaaac ctcttcgaggcacaaggcac aacaggctgc tctgggattc 1980 tcttcagcca atcttcattg ctcaagtatgactttaatct tccttacaac taggtgctaa 2040 gggagtctct ctgtctctct gcctctttgtgtgtatgcat attctctctc tctctctctt 2100 tctttctctg tctctcctct ccttcctctctgcctcctct ctcagctttt tgcaaaaatg 2160 ccaggtgtaa tataatgctt atgactcgggaaatattctg ggaatggata ctgcttatct 2220 aacagctgac accctaaagg ttagtgtcaaagcctctgct ccagctctcc tagccaatac 2280 attgctagtt ggggtttggt ttagcaaatgcttttctcta gacccaaagg acttctcttt 2340 cacacattca ttcatttact cagagatcatttctttgcat gactgccatg cactggatgc 2400 tgagagaaat cacacatgaa cgtagccgtcatggggaagt cactcatttt ctccttttta 2460 cacaggtgtc tgaagcagcc atggcagaagtacctgagct cgccagtgaa atgatggctt 2520 attacaggtc agtggagacg ctgagaccagtaacatgagc aggtctcctc tttcaagagt 2580 agagtgttat ctgtgcttgg agaccagatttttcccctaa attgcctctt tcagtggcaa 2640 acagggtgcc aagtaaatct gatttaaagactactttccc attacaagtc cctccagcct 2700 tgggacctgg aggctatcca gatgtgttgttgcaagggct tcctgcagag gcaaatgggg 2760 agaaaagatt ccaagcccac aatacaaggaatccctttgc aaagtgtggc ttggagggag 2820 agggagagct cagattttag ctgactctgctgggctagag gttaggcctc aagatccaac 2880 agggagcacc agggtgccca cctgccaggcctagaatctg ccttctggac tgttctgcgc 2940 atatcactgt gaaacttgcc aggtgtttcaggcagctttg agaggcaggc tgtttgcagt 3000 ttcttatgaa cagtcaagtc ttgtacacagggaaggaaaa ataaacctgt ttagaagaca 3060 taattgagac atgtccctgt ttttattacagtggcaatga ggatgacttg ttctttgaag 3120 ctgatggccc taaacagatg aaggtaagactatgggttta actcccaacc caaggaaggg 3180 ctctaacaca gggaaagctc aaagaagggagttctgggcc actttgatgc catggtattt 3240 tgttttagaa agactttaac ctcttccagtgagacacagg ctgcaccact tgctgacctg 3300 gccacttggt catcatatca ccacagtcactcactaacgt tggtggtggt ggccacactt 3360 ggtggtgaca ggggaggagt agtgataatgttcccatttc atagtaggaa gacaaccaag 3420 tcttcaacat aaatttgatt atccttttaagagatggatt cagcctatgc caatcacttg 3480 agttaaactc tgaaaccaag agatgatcttgagaactaac atatgtctac cccttttgag 3540 tagaatagtt ttttgctacc tggggtgaagcttataacaa caagacatag atgatataaa 3600 caaaaagatg aattgagact tgaaagaaaaccattcactt gctgtttgac cttgacaagt 3660 cattttaccc gctttggacc tcatctgaaaaataaagggc tgagctggat gatctctgag 3720 attccagcat cctgcaacct ccagttctgaaatattttca gttgtagcta agggcatttg 3780 ggcagcaaat ggtcattttt cagactcatccttacaaaga gccatgttat attcctgctg 3840 tcccttctgt tttatatgat gctcagtagccttcctaggt gcccagccat cagcctagct 3900 aggtcagttg tgcaggttgg aggcagccacttttctctgg ctttatttta ttccagtttg 3960 tgatagcctc ccctagcctc ataatccagtcctcaatctt gttaaaaaca tatttcttta 4020 gaagttttaa gactggcata acttcttggctgcagctgtg ggaggagccc attggcttgt 4080 ctgcctggcc tttgcccccc attgcctcttccagcagctt ggctctgctc caggcaggaa 4140 attctctcct gctcaacttt cttttgtgcacttacaggtc tctttaactg tctttcaagc 4200 ctttgaacca ttatcagcct taaggcaacctcagtgaagc cttaatacgg agcttctctg 4260 aataagagga aagtggtaac atttcacaaaaagtactctc acaggatttg cagaatgcct 4320 atgagacagt gttatgaaaa aggaaaaaaaagaacagtgt agaaaaattg aatacttgct 4380 gagtgagcat aggtgaatgg aaaatgttatggtcatctgc atgaaaaagc aaatcatagt 4440 gtgacagcat tagggataca aaaagatatagagaaggtat acatgtatgg tgtaggtggg 4500 gcatgtacaa aaagatgaca agtagaatcgggatttattc taaagaatag cctgtaaggt 4560 gtccagaagc cacattctag tcttgagtctgcctctacct gctgtgtgcc cttgagtaca 4620 cccttaacct ccttgagctt cagagagggataatcttttt attttatttt attttatttt 4680 gttttgtttt gttttgtttt gttttatgagacagagtctc actctgttgc ccaggctgga 4740 gtgcagtggt acaatcttgg cttactgcatcctccacctc ctgagttcaa gcgattctcc 4800 ttcctcagtc tcctgaatag ctaggattacaggtgcaccc caccacaccc agctaatttt 4860 tgtattttta gtagagaagg ggtttcgccatgttggccag gctggttttg aagtcctgac 4920 ctaaatgatt catccacctc ggcttcccaaagtgctggga ttacaggcat gagccaccac 4980 gcctggccca gagagggatg atctttagaagctcgggatt ctttcaagcc ctttcctcct 5040 ctctgagctt tctactctct gatgtcaaagcatggttcct ggcaggacca cctcaccagg 5100 ctccctccct cgctctctcc gcagtgctccttccaggacc tggacctctg ccctctggat 5160 ggcggcatcc agctacgaat ctccgaccaccactacagca agggcttcag gcaggccgcg 5220 tcagttgttg tggccatgga caagctgaggaagatgctgg ttccctgccc acagaccttc 5280 caggagaatg acctgagcac cttctttcccttcatctttg aagaaggtag ttagccaaga 5340 gcaggcagta gatctccact tgtgtcctcttggaagtcat caagccccag ccaactcaat 5400 tcccccagag ccaaagccct ttaaaggtagaaggcccagc ggggagacaa aacaaagaag 5460 gctggaaacc aaagcaatca tctctttagtggaaactatt cttaaagaag atcttgatgg 5520 ctactgacat ttgcaactcc ctcactctttctcaggggcc tttcacttac attgtcacca 5580 gaggttcgta acctccctgt gggctagtgttatgaccatc accattttac ctaagtagct 5640 ctgttgctcg gccacagtga gcagtaatagacctgaagct ggaacccatg tctaatagtg 5700 tcaggtccag tgttcttagc caccccactcccagcttcat ccctactggt gttgtcatca 5760 gactttgacc gtatatgctc aggtgtcctccaagaaatca aattttgcca cctcgcctca 5820 cgaggcctgc ccttctgatt ttatacctaaacaacatgtg ctccacattt cagaacctat 5880 cttcttcgac acatgggata acgaggcttatgtgcacgat gcacctgtac gatcactgaa 5940 ctgcacgctc cgggactcac agcaaaaaagcttggtgatg tctggtccat atgaactgaa 6000 agctctccac ctccagggac aggatatggagcaacaaggt aaatggaaac atcctggttt 6060 ccctgcctgg cctcctggca gcttgctaattctccatgtt ttaaacaaag tagaaagtta 6120 atttaaggca aatgatcaac acaagtgaaaaaaaatatta aaaaggaata tacaaacttt 6180 ggtcctagaa atggcacatt tgattgcactggccagtgca tttgttaaca ggagtgtgac 6240 cctgagaaat tagacggctc aagcactcccaggaccatgt ccacccaagt ctcttgggca 6300 tagtgcagtg tcaattcttc cacaatatggggtcatttga tggacatggc ctaactgcct 6360 gtgggttctc tcttcctgtt gttgaggctgaaacaagagt gctggagcga taatgtgtcc 6420 atccccctcc ccagtcttcc ccccttgccccaacatccgt cccacccaat gccaggtggt 6480 tccttgtagg gaaattttac cgcccagcaggaacttatat ctctccgctg taacgggcaa 6540 aagtttcaag tgcggtgaac ccatcattagctgtggtgat ctgcctggca tcgtgccaca 6600 gtagccaaag cctctgcaca ggagtgtgggcaactaaggc tgctgacttt gaaggacagc 6660 ctcactcagg gggaagctat ttgctctcagccaggccaag aaaatcctgt ttctttggaa 6720 tcgggtagta agagtgatcc cagggcctccaattgacact gctgtgactg aggaagatca 6780 aaatgagtgt ctctctttgg agccactttcccagctcagc ctctcctctc ccagtttctt 6840 cccatgggct actctctgtt cctgaaacagttctggtgcc tgatttctgg cagaagtaca 6900 gcttcacctc tttcctttcc ttccacattgatcaagttgt tccgctcctg tggatgggca 6960 cattgccagc cagtgacaca atggcttccttccttccttc cttcagcatt taaaatgtag 7020 accctctttc attctccgtt cctactgctatgaggctctg agaaaccctc aggcctttga 7080 ggggaaaccc taaatcaaca aaatgaccctgctattgtct gtgagaagtc aagttatcct 7140 gtgtcttagg ccaaggaacc tcactgtgggttcccacaga ggctaccaat tacatgtatc 7200 ctactctcgg ggctaggggt tggggtgaccctgcatgctg tgtccctaac cacaagaccc 7260 ccttctttct tcagtggtgt tctccatgtcctttgtacaa ggagaagaaa gtaatgacaa 7320 aatacctgtg gccttgggcc tcaaggaaaagaatctgtac ctgtcctgcg tgttgaaaga 7380 tgataagccc actctacagc tggaggtaagtgaatgctat ggaatgaagc ccttctcagc 7440 ctcctgctac cacttattcc cagacaattcaccttctccc cgcccccatc cctaggaaaa 7500 gctgggaaca ggtctatttg acaagttttgcattaatgta aataaattta acataatttt 7560 taactgcgtg caaccttcaa tcctgctgcagaaaattaaa tcattttgcc gatgttatta 7620 tgtcctacca tagttacaac cccaacagattatatattgt tagggctgct ctcatttgat 7680 agacaccttg ggaaatagat gacttaaagggtcccattat cacgtccact ccactcccaa 7740 aatcaccacc actatcacct ccagctttctcagcaaaagc ttcatttcca agttgatgtc 7800 attctaggac cataaggaaa aatacaataaaaagcccctg gaaactaggt acttcaagaa 7860 gctctagctt aattttcacc cccccaaaaaaaaaaaattc tcacctacat tatgctcctc 7920 agcatttggc actaagtttt agaaaagaagaagggctctt ttaataatca cacagaaagt 7980 tgggggccca gttacaactc aggagtctggctcctgatca tgtgacctgc tcgtcagttt 8040 cctttctggc caacccaaag aacatctttcccataggcat ctttgtccct tgccccacaa 8100 aaattcttct ttctctttcg ctgcagagtgtagatcccaa aaattaccca aagaagaaga 8160 tggaaaagcg atttgtcttc aacaagatagaaatcaataa caagctggaa tttgagtctg 8220 cccagttccc caactggtac atcagcacctctcaagcaga aaacatgccc gtcttcctgg 8280 gagggaccaa aggcggccag gatataactgacttcaccat gcaatttgtg tcttcctaaa 8340 gagagctgta cccagagagt cctgtgctgaatgtggactc aatccctagg gctggcagaa 8400 agggaacaga aaggtttttg agtacggctatagcctggac tttcctgttg tctacaccaa 8460 tgcccaactg cctgccttag ggtagtgctaagaggatctc ctgtccatca gccaggacag 8520 tcagctctct cctttcaggg ccaatccccagcccttttgt tgagccaggc ctctctcacc 8580 tctcctactc acttaaagcc cgcctgacagaaaccacggc cacatttggt tctaagaaac 8640 cctctgtcat tcgctcccac attctgatgagcaaccgctt ccctatttat ttatttattt 8700 gtttgtttgt tttgattcat tggtctaatttattcaaagg gggcaagaag tagcagtgtc 8760 tgtaaaagag cctagttttt aatagctatggaatcaattc aatttggact ggtgtgctct 8820 ctttaaatca agtcctttaa ttaagactgaaaatatataa gctcagatta tttaaatggg 8880 aatatttata aatgagcaaa tatcatactgttcaatggtt ctgaaataaa cttcactgaa 8940 gaaaaaaaaa aaagggtctc tcctgatcattgactgtctg gattgacact gacagtaagc 9000 aaacaggctg tgagagttct tgggactaagcccactcctc attgctgagt gctgcaagta 9060 cctagaaata tccttggcca ccgaagactatcctcctcac ccatcccctt tatttcgttg 9120 ttcaacagaa ggatattcag tgcacatctggaacaggatc agctgaagca ctgcagggag 9180 tcaggactgg tagtaacagc taccatgatttatctatcaa tgcaccaaac atctgttgag 9240 caagcgctat gtactaggag ctgggagtacagagatgaga acagtcacaa gtccctcctc 9300 agataggaga ggcagctagt tataagcagaacaaggtaac atgacaagta gagtaagata 9360 gaagaacgaa gaggagtagc caggaaggagggaggagaac gacataagaa tcaagcctaa 9420 agggataaac agaagatttc cacacatgggctgggccaat tgggtgtcgg ttacgcctgt 9480 aatcccagca ctttgggtgg caggggcagaaagatcgctt gagcccagga gttcaagacc 9540 agcctgggca acatagtgag actcccatctctacaaaaaa taaataaata aataaaacaa 9600 tcagccaggc atgctggcat gcacctgtagtcctagctac ttgggaagct gacactggag 9660 gattgcttga gcccagaagt tcaagactgcagtgagctta tccgttgacc tgcaggtcga 9720 c 9721 3 12565 DNA Homo sapiens 3gtcgacctgc aggtcaacgg atctgagagg agagtagctt cttgtagata acagttggat 60tatataccat gtcctgatcc ccttcatcat ccaggagagc agaggtggtc accctgatag 120cagcaagcct gggggctgca gcttggtggg tagaggtact caggggtaca gatgtctcca 180aacctgtcct gctgccttag ggagcttcta ataagttgat ggatttggtt aaaattaact 240tggctacttg gcaggactgg gtcagtgagg accaacaaaa agaagacatc agattatacc 300ctgggggttt gtatttcttg tgtttctttc tcttctttgt actaaaatat ttacccatga 360ctgggaaaga gcaactggag tctttgtagc attatcttag caaaaattta caaagtttgg 420aaaacaatat tgcccatatt gtgtggtgtg tcctgtgaca ctcaggattc aagtgttggc 480cgaagccact aaatgtgaga tgaagccatt acaaggcagt gtgcacatct gtccacccaa 540gctggatgcc aacatttcac aaatagtgct tgcgtgacac aaatgcagtt ccaggaggcc 600caaatgaaaa tgtttgtact gaaatttgtt aaagcttccc gacaaactag atttatcagt 660aaggattgtt ttctgcaagg gggatgaaac ttgtggggtg agccatttgg gctgaggagg 720agggaggttg gagctgagaa atgtggagac aatttccctt tagaaggact gaatctccct 780gcctctctgg ggtgcggcag ccagcaggat ccaatggtgt atatgtctcc ccagctcccc 840attcagtgat atcatgtcag tagcttgaaa ttatccgtgg tgggagtatt atgtcatgga 900aattggcaaa tggaaacttt tattggagat tcaattgtta aacttttacc agcacaacac 960tgccctgcct tcagagtcaa tgaccctatc caagtttaat ccatctgtcc actgtctcca 1020acacgatctt tataaaacac acctgacaac attacccttt tattcagttt tttaaaagat 1080aagtttccag ctcatcgggg tggctttaaa ggccatttct cctctggacc tcacccaact 1140tttcaaatca cttttcctac ccctacctct aaatgctact caaactccag ccatcctgaa 1200taataagact tttgaaaagt agattatggg ctgggcacag tggctcacac ctgtaatccc 1260agcactttgg gaggccaaga tgggtggatc acctgaggtc gggagttcga gaccagcctg 1320actaacatag tgaaaccctg tctctactaa aaatacaaaa ttagttgggg gtggtggcac 1380aagcctgtaa tcccagctac tcaggaggtt gaggcagggg aattgcttga acctgggagg 1440cggaggttgc ggtgagccta gattgctcca ctgcactcca gcctgggcaa caagagcgaa 1500actccatctc aaaaaaataa ataaataaat aaagtagatt acatcagata cctctggcct 1560aggttgttta tgaccaactc tcctgctgag aataactaga aaagctagac aaaacatatt 1620tccaaaagat ctctttggag gcatcagaga atggccaagg ctgtaaggaa ctgcctgagc 1680ccagagaggt ggagcccagc actggtgccc tttactcctg gggacatgtg ctggtttcaa 1740aaacttcagc tgagcttttg agcattcatg gaacttggtg ggggagatga aatttgtacc 1800ttaaatcctg cctacaggga gggtccctga taatccccac ccaatttgga aatctgggtc 1860agccttcaca ggtactgaag ccctcctctg aatgatctca agtcctgcta gggtagaggt 1920tacctgcttt tgaaaggctc ctggcctacc tgtgcagcag gagcaaaagt gaaccatctc 1980agggtacaga taacaatcat ccagagcctt gaatgacctc tactgtgctt aatatatagt 2040attcagcagt cagtaaaaag gatttaggca catgcaagat gacctgtgta tcagggagaa 2100ataggcaata aattgagatc cagcagggat ttgaatcatg gatttgaatc aggggcagcc 2160ttcgaaagaa ctatggagaa tatactcaga tttaaaacat aagattggaa tttttggcag 2220agaactaaca actgtacaaa aaaggaacca aatggaaatc ctagaactga aagatgcaat 2280taaccgatgt tgagaaatag ccaacatcta ttgaacactt cccatgtgga cagctgtgct 2340aaacacttta caggcatcaa cataagatgt gtccccttac agcagtgcag tgtccctcct 2400aagacatgga cagcctggtt tccctatctc tctgcttcat caaaacccct ttacgtgggg 2460cttagacact cctgttgtct ctagtgtcta gtagcacagg gctcagcaca tggaagccac 2520tagatacaat ttgatgacca ggacctccga tgaaagccat gggtgctgat tgggaaggca 2580ttgtctttta tgtgctatgg tcttaaagct tcatccagga agcagaactc ggggggtgct 2640gaggacccag aaccgagaat aagattagtc agagatttcc tgtgggcaga aatcataagg 2700acgccaactg tttgggtgag ataagacgaa accaagagtg gacttgtggc cagaagcgtg 2760aggaagaggg agagagcttc ccttgtcccc tttcttcctc tccctaagcc acagtgattg 2820acagcccccc cgctttggag tcagagcagg cttgagactg gactgggaaa ggagggtggg 2880tcaggataca gagcaggaag gctgggagtg cagggcagga gcaaggggct ggggcattca 2940ttgtgcctga tctctcccac tttacctggg gtaaagaagc atatgcaaaa gccacggtgt 3000gagtatttcc caagtgccag ggtcagggca tgattcatca cgtgcagcat ttcattcaat 3060ccttatagta accgatgatg tggcttctat tattagctct atcagataat gaaactgaga 3120ccaagacagg ctctgcacat tgtgtggggt aatgacacag ggggattcag acctagactc 3180cataactcct gccccaggga ccacccccac cctcaccctg tgcatgtcga caaaggacag 3240actgggccac ttctcaggac acagcgggga aatgacacag agcagggagg ttccaggagc 3300cccgagcgtc ttttctccag gagaatactc tctgaattca gactggggtc agagaaacat 3360ttacccagga gccgcagtgt gggtggggct ttttacttga aacgctgtct gaaggcagtg 3420gcaggatgaa ctctccaccc taccttggca agccacttct cttctgcaat ctgtaaggac 3480attgttgaga gaattatggt cttccaattc cggagggttg aagaaagaca aataggagag 3540aacctatcat agtcaggtgc tagctgcctt ctctttcaga gagtgtgaga ataaagtgat 3600acacttgatt attagcaaat actttggaaa ttttaaacgc taatattcaa cacactctgg 3660aagaggcaaa taagtagaca ggttcatata catcatctcc ttcagctagt cctcacaaaa 3720acaaacaaat gaataaacaa aattcttctt tggccctcat aggaagacac tgtttcttga 3780acgtgtttca aaaaggatgg gtgactcact caaggtcaca ctgtttatga ggacagtaca 3840ggaatacaga catgccattt tgcctgaaaa aatccatcac ccagggaggt gacacaattt 3900tgcagaaatg ttctatttcc tctgaaggat acattcttta aacctttggg aaattcattc 3960atagtcttcc tcctttgaag gattactctc tggacacaaa gtgtttgatt ctgatttgtt 4020ggttggaaga tgtgttggtt gagagaaaga ttctgatttg ttggttgaaa atagactcat 4080caagatcaac tgctgtagta gtaaatattt tgacattttg tctgtattcc tgtgctgccc 4140tcacaagctg catcaccttg agtgagtcat tcatactttt ttgtttgttt ttgttttgga 4200gatggagtct tactctgttg cctaggctgg agtgcggtgg cgtgatcttg gctcactgcg 4260acctccatct cctgggttca agtgatcctc ctgcctcagc ctcccgagta gctgggatta 4320caggcacatg ccaccatccc tgctaatttt tgcattttca gtagagacgg agtttcacca 4380tgttggtcag gttggtcttg aactcctgac ctcaggtgat ccgcccacct cagcctcccc 4440aagtgctggg attacaggtg tgagccaccg tgcccagccc agccatcatt tttgaaacac 4500gtttgagaaa tagtgtcttc ctttgagggc caaggagaca ttttttttgt ttatttgttt 4560gtttttgtga ggactagctg aagggggtga tgtatattaa cctgcctact tatttgcctc 4620ttcccagagt gtgatgaata ttagggttta aagtttctga agcatttgtt aataaagccc 4680ggggctggag gtcagaagac ctggatttct ctgcatactt ttgccatcag caagctgtgt 4740gaccttggac agatcccttt tttgtctaaa tctttctgag tcttcttgaa aacaatgcca 4800ggttgggaca ggatgattgc caagctcccg tccagctcta aaacactgca acgtatgctt 4860ctgcaccagc actgtccatc ctgtagatca tgcagaaatt ctcttcaact ttttcctacc 4920cataaaatag gagcatgctt acctttttcc taatgttcca ggccccgggt ctagatattg 4980taagtaagga agttaatgtg tatcagagcc cattatgggc cagaagttct cctcttcctt 5040cctacacctg cttcctccct ccctccctcc ctctttccct tccttccttc catccatttg 5100tgaagaagac atgatcaccc tcattctgag agtgaagaga cagaggctca actaatgaaa 5160tgatttgttc aaggtcacac gggtggcaca aggcaagtgg cagaggttga atttagaccc 5220attcctgtcc aaatgctgag tttatgtcat cgtcccgaga ccataacttt aaagatgtaa 5280gatagtggga aaagagttga tttcaaagca cctctcagaa ggactcactt tacatcaggg 5340gtcagcagac tcaggccaaa tccggtccat tccccgcttt tgcaaagaaa gttgtagtgg 5400aacacagcta ggcttattga tttatggatt gccaacgtcc ttttgtgaaa cagacagctg 5460agctgagtaa tcgtggcgca caaaacctaa aatatttact atctcgtcct ttacagaatg 5520tttgccaatc tatggtccgg agtccaaggc tgtccatttt tcaaagaaca caaagtgaca 5580tgagactgtc ccatgtgcag ggagccctat cattttatta tgaaaaaacg gcctttctgc 5640tcaaatctgt tttttaaaaa gtcaacaaac agactctggg tacctgtcag gaacagtagg 5700gagtttggtt tccattgtgc tcttcttccc aggaactcaa tgaaggggaa atagaaatct 5760taattttggg gaaattgcac aggggaaaaa ggggagggaa tcagttacaa cactccattg 5820cgacacttag tggggttgaa agtgacaaca gcaagggttt ctctttttgg aaatgcgagg 5880agggtatttc cgcttctcgc agtggggcag ggtggcagac gcctagcttg ggtgagtgac 5940tatttcttta taaaccacaa ctctgggccc gcaatggcag tccactgctt gctgcagtca 6000cagaatggaa atctgcagag gcctccgcag tcacctaatc actctcctcc tcttcctgtt 6060ccattcagag acgatctgcc gaccctctgg gagaaaatcc agcaagatgc aagccttcag 6120gtaaggctac cccaaggagg agaaggtgag ggtggatcag ctggagactg gaaacatatc 6180acagctgcca gggctgccag gccagagggc ctgagaactg ggtttgggct ggagaggatg 6240tccattattc aagaaagagg ctgttacatg catgggcttc aggacttgtg tttcaaaata 6300tcccagatgt ggatagtgcg accggagggc tgtcttactt tcccagagac tcaggaaccc 6360agtgagtaat agatgcatgc caaggagtgg gactgcgatt caggcctagt tgaatgtgct 6420gacagagaag cagagagggg caccaggggc acagcccgaa ggcccagact gatatgggca 6480aggcctgtct gtgctgacat gtcggagggt cccactctcc agggaccttg gtttccccgt 6540ctgtgacatc tgtgacatga gagtcacgat aactccttgt gtgccttaca gggttgttgt 6600gaaaattaaa tgcacagata atagcgtaac agtattccgt gcattgtaaa gagcctgaaa 6660accattatga tttgaaaatg gaatcggctt tgtgagacca tcactattgt aaagatgtga 6720tgctgataga aatgacagga ctgcttgtgc atgccctctg cagtgtgaca ttccagcagt 6780gaaatcatgt tggggtgact tctcccccac tctgaccttt atgtttgtct gggccgaggc 6840tgcaagtcgg gctctgtggg tgtatgagtg acaagtctct cccttccaga tatggggact 6900gtctgcttcc ctaggttgcc tctccctgct ctgatcagct agaagctcca ggagatcctc 6960ctggaggccc cagcaggtga tgtttatccc tccagactga ggctaaatct agaaactagg 7020ataatcacaa acaggccaat gctgccatat gcaaagcact ttggtttgcc tggccacccc 7080tcgtcgagca tgtgggctct tcagagcacc tgatgaggtg ggtacagtta gccacacttc 7140acaggtgaag aggtgaggca caggtcccag gtcaggctgg ccggagctct gtttattacg 7200tctcacagct ttgagtcctg ctctcaacca gagaggccct ttaccaagaa gaaaggattg 7260ggacccagaa tcaggtcact ggctgaggta gagaggaagc cgggttgttc ccaagggtag 7320ctgctcctgc aggactctga gcaggtcacc agctaatgga ggaaaggctc tagggaaaga 7380cccttctggt ctcagactca gagcgagtta gctgcaaggt gttccgtctc ttgaaacttc 7440tacctaggtg ctatggtagc cactagtctc aggtggctat ttaaatttat acttaaatga 7500atgaaaatag aagaaaattt aaaatccaga cccttggtca cactatccac atttaaagag 7560gtcaatagcc acatgtggtt agtggccacc ctattgggca gtgcagctac agaacatttt 7620tgcatcccag aaagttcttt tggatgttgc tgctctacag catgctttgc tgaaacagaa 7680gtgccttccc tgggaatctc agatgggaag caagtaagga ggggagtcaa atgtgggctc 7740actgctcacc agctgtgagg gttgggcctg cctcttaacc attgtcagcc tcagtcttct 7800catccatgca tgccgtgggt atactaaaat actatacccc tggaagagct ggatgcaaat 7860ttgacaagtt ctgggggaca caggaaggtg ccaagcacaa ggctgggcac atggtggctg 7920tgcactacag ctgagtcctt ttccttttca gaatctggga tgttaaccag aagaccttct 7980atctgaggaa caaccaacta gttgctggat acttgcaagg accaaatgtc aatttagaag 8040gtgagtggtt gccaggaaag ccaatgtatc tgggcatcac gtcactttgc ccgtctgtct 8100gcagcagcat ggcctgcctg cacaaaccct aggtgcaatg tcctaatcct tgttgggtct 8160ttgtattcaa gtttgaagct gggagggcct ggctactgaa gggcacatat gagggtagcc 8220tgaagagggt gtggagaggt agagtctagg tcagaggtca gtgcctatag gcaagtggtc 8280ccagggccac agctgggaag ggcaaatacc agaaggcaag gttgaccatt cccttcctca 8340agtgcctatt aaggctccat gttcctatgt tgttcaaacc ctaactcaat cccaaattaa 8400tccaccatgt ataaggttga gctatgtctc ttattcctgg acaccatact cagccatatc 8460tggtccacac attaacagct ggatgacctt gaagaagctt cacccactct gttcctcagc 8520tttcccttca gtgggatgat atcaactgga caacaggatg tgcgattctt ttagttccag 8580ccttccagga tgttttcact cccctgtttg ttgttgtagg atggtattac ctccaccttc 8640ccaccttccc tatgccctgg ttctgtctcc tgtgcctcgc tctgaaagtg gatgagacct 8700acaattcctg tcctggtagt tctcctaatg aacacactga agcacgagga agctgagatt 8760tttgttgcta catgagagca tggaggcctc ttagggagag aggaggttca gagactccta 8820ggctcctggt ggagccccac tcatggcctt gttcattttc cctgcccctc agcaacactc 8880ctattgacct ggagcacagg tatcctgggg aaagtgaggg aaatatggac atcacatgga 8940acaacatcca ggagactcag gcctctagga gtaactgggt agtgtgcatc ctggggaaag 9000tgagggaaat atggacatca catggaacaa catccaggag actcaggcct ctaggagtaa 9060ctgggtagtg tgcatcctgg ggaaagtgag ggaaatatgg acatcacatg gaacaacatc 9120caggagactc aggcctctag gagtaactgg gtagtgtgca tcctggggaa agtgagggaa 9180atatggacat cacatggaac aacatccagg agactcaggc ctctaggagt aactgggtag 9240tgtgcttggt ttaatcttct atttacctgc agaccaggaa gatgagacct ctctgccctt 9300ctgacctcgg gattttagtt ttgtggggac caggggagat agaaaaatac ccggggtctc 9360ttcattattg ctgcttcctc ttctattaac ctgaccctcc cctctgttct tccccagaaa 9420agatagatgt ggtacccatt gagcctcatg ctctgttctt gggaatccat ggagggaaga 9480tgtgcctgtc ctgtgtcaag tctggtgatg agaccagact ccagctggag gtaaaaacat 9540gctttggatc tcaaatcacc ccaaaaccca gtggcttgaa acaaccaaaa ttttttctta 9600tgattctgtg ggttgaccag gattagctgg gtagttctgt tccatgtggt ggaacatgct 9660ggggtcactt tggaagctgc attcagcaga gtgccaggct tgcgctgggc atccaaggtg 9720gtccctcatc ctccaggctc tctttccatg tgatctctca gtgtttaaga gttagttgga 9780gcttccttac agcatggcgg ctgacttcca aaagggatta ttccaaaaag agcctcaaca 9840tgcaggcgct tattatgact tctgcttgca tcatcctatt ggccaaagcc agtcacgtgg 9900ctaagtctag ccccctgtga gaggagactg cataagagtg tgaacaccag gagacacggt 9960cactgggggc caccactgta accatctacc acaggacctg aatctctgtg tgctactccc 10020ttgctcaagg gcccccctac ccacgcagac ctgctgtctt ctagcaaagc ccatcctcag 10080gacctttctc ttccaatcct tattgactca aattgattag ttggtgctcc acccagagcc 10140ctgtgctcct ttatctcatg taatgttaat gggtttccca gccctgggaa aacatggctt 10200tgtctcaggg gcttgctgga tgcaacctta acctcaatgt gagtggccat actgtggcac 10260tgtcccatcc ctcaccaggg acactgttct ggagggtgac tgcctgttct gtgaggagtg 10320gggatggcta ggacattgca tggaacacac caccacccca tcttctcaga gctcaaaccc 10380tgacagaaca ccagctccac aggccttggc ttctgctgat ggtgccgtgt atttaccaga 10440cttagtggtc caaggccaga gtggcagatt tcccaaagtc aaggtgtgac agtgggacag 10500cctctttgtg tctttgctgt cctaagaaac ctgggccagg ccaggcgcag tggctcacgc 10560cttgtaatcc cagcactttg agaggccaag gtgggcagat cacgaggtca ggagtttgag 10620accagcctgg ccaacattgg tgaaaccctg tctctattaa aaatagaaaa cattagacag 10680gtgtggtggt gcatgcctgt aatcccagct actcaggagg ctgaggcagg agaatcgctt 10740gaacccagga ggtggaggtt gcagtgagcc gagattgtgc cactgcactc cagcctaggc 10800gacagagcaa gactccgtct cgggaaaatt aattaataaa taaataaacc taggtcccag 10860agtcccacag aatggcagac aggagcacct gggggctttt agggtatggc atttcccctg 10920tactaactct gggctgtcca gaggcgattt catggcgtgg agtggagagg gaggcagcac 10980aggacttcct aggcctcagc tctcacctgc ccatcttttg atttccaggc agttaacatc 11040actgacctga gcgagaacag aaagcaggac aagcgcttcg ccttcatccg ctcagacagt 11100ggccccacca ccagttttga gtctgccgcc tgccccggtt ggttcctctg cacagcgatg 11160gaagctgacc agcccgtcag cctcaccaat atgcctgacg aaggcgtcat ggtcaccaaa 11220ttctacttcc aggaggacga gtagtactgc ccaggcctgc ctgttcccat tcttgcatgg 11280caaggactgc agggactgcc agtccccctg ccccagggct cccggctatg ggggcactga 11340ggaccagcca ttgaggggtg gaccctcaga aggcgtcaca acaacctggt cacaggactc 11400tgcctcctct tcaactgacc agcctccatg ctgcctccag aatggtcttt ctaatgtgtg 11460aatcagagca cagcagcccc tgcacaaagc ccttccatgt cgcctctgca ttcaggatca 11520aaccccgacc acctgcccaa cctgctctcc tcttgccact gcctcttcct ccctcattcc 11580accttcccat gccctggatc catcaggcca cttgatgacc cccaaccaag tggctcccac 11640accctgtttt acaaaaaaga aaagaccagt ccatgaggga ggtttttaag ggtttgtgga 11700aaatgaaaat taggatttca tgattttttt ttttcagtcc ccgtgaagga gagcccttca 11760tttggagatt atgttctttc ggggagaggc tgaggactta aaatattcct gcatttgtga 11820aatgatggtg aaagtaagtg gtagcttttc ccttcttttt cttctttttt tgtgatgtcc 11880caacttgtaa aaattaaaag ttatggtact atgttagccc cataattttt tttttccttt 11940taaaacactt ccataatctg gactcctctg tccaggcact gctgcccagc ctccaagctc 12000catctccact ccagattttt tacagctgcc tgcagtactt tacctcctat cagaagtttc 12060tcagctccca aggctctgag caaatgtggc tcctgggggt tctttcttcc tctgctgaag 12120gaataaattg ctccttgaca ttgtagagct tctggcactt ggagacttgt atgaaagatg 12180gctgtgcctc tgcctgtctc cccaccaggc tgggagctct gcagagcagg aaacatgact 12240cgtatatgtc tcaggtccct gcagggccaa gcacctagcc tcgctcttgg caggtactca 12300gcgaatgaat gctgtatatg ttgggtgcaa agttccctac ttcctgtgac ttcagctctg 12360ttttacaata aaatcttgaa aatgcctata ttgttgacta tgtccttggc cttgacaggc 12420tttgggtata gagtgctgag gaaactgaaa gaccaatgtg tyttycttac cccagaggct 12480ggcgcctggc ctcttctctg agagttcttt tcttccttca gcctcactct ccctggataa 12540catgagagca aatctctctg cgggg 12565 4 25 DNA Artificial SequenceDescription of Artificial Sequence primer 4 tgtacctaag cccacccttt agagc25 5 20 DNA Artificial Sequence Description of Artificial Sequenceprimer 5 tggcctccag aaacctccaa 20 6 20 DNA Artificial SequenceDescription of Artificial Sequence primer 6 gctgatattc tggtgggaaa 20 720 DNA Artificial Sequence Description of Artificial Sequence primer 7ggcaagagca aaactctgtc 20 8 21 DNA Artificial Sequence Description ofArtificial Sequence primer 8 atgtatagaa ttccattcct g 21 9 21 DNAArtificial Sequence Description of Artificial Sequence primer 9taaaatcaag tgttgatgta g 21 10 24 DNA Artificial Sequence Description ofArtificial Sequence primer 10 gggatacagg cgtgagccac cgcg 24 11 25 DNAArtificial Sequence Description of Artificial Sequence primer 11ttagtattgc tggtagtatt catat 25 12 21 DNA Artificial Sequence Descriptionof Artificial Sequence primer 12 tgttctacca cctgaactag g 21 13 20 DNAArtificial Sequence Description of Artificial Sequence primer 13ttacatatga gccttccatg 20 14 25 DNA Artificial Sequence Description ofArtificial Sequence primer 14 ctcaggtgtc ctcgaagaaa tcaaa 25 15 19 DNAArtificial Sequence Description of Artificial Sequence primer 15gctttmgctg tgagtcccg 19 16 20 DNA Artificial Sequence Description ofArtificial Sequence primer 16 tggcattgat ctggttcatc 20 17 20 DNAArtificial Sequence Description of Artificial Sequence primer 17gtttaggaat cttcccactt 20 18 20 DNA Artificial Sequence Description ofArtificial Sequence primer 18 gaggcgtgag aatctcaaga 20 19 20 DNAArtificial Sequence Description of Artificial Sequence primer 19gtgtcctcaa gtggatctgg 20 20 21 DNA Artificial Sequence Description ofArtificial Sequence primer 20 gggcaacaga gcaatgtttc t 21 21 20 DNAArtificial Sequence Description of Artificial Sequence primer 21cagtgtgtca gtgtactgtt 20 22 17 DNA Artificial Sequence Description ofArtificial Sequence primer 22 ctcagcaaca ctcctat 17 23 17 DNA ArtificialSequence Description of Artificial Sequence primer 23 tcctggtctg caggtaa17 24 24 DNA Artificial Sequence Description of Artificial Sequenceprimer 24 ttacgcagat aagaaccagt ttgg 24 25 22 DNA Artificial SequenceDescription of Artificial Sequence primer 25 tttcctggac gcttgctcac ca 2226 29 DNA Artificial Sequence Description of Artificial Sequence primer26 ttctatctga ggaacaacca actagtagc 29 27 29 DNA Artificial SequenceDescription of Artificial Sequence primer 27 caccagactt gacacaggacaggcacatc 29 28 27 DNA Artificial Sequence Description of ArtificialSequence primer 28 cgaccctctg ggagaaaatc cagcaag 27 29 20 DNA ArtificialSequence Description of Artificial Sequence primer 29 acacaggaaggtgccaagca 20 30 20 DNA Artificial Sequence Description of ArtificialSequence primer 30 tgcagacaga cgggcaaagt 20 31 20 DNA ArtificialSequence Description of Artificial Sequence primer 31 ttgtggggaccaggggagat 20 32 20 DNA Artificial Sequence Description of ArtificialSequence primer 32 agcctggcac tctgctgaat 20

What is claimed is:
 1. A kit comprising a means for detecting one or more alleles selected from the group consisting of: allele 4 of the 222/223 marker of IL-1A, allele 4 of the gz5/gz6 marker of IL-1A, allele 1 of the −889 marker of IL-1A, allele 1 of the +3954 marker of IL-1B, allele 2 of the −511 marker of IL-1B, allele 3 of the gaat.p33330 marker, allele 3 of the Y31 marker, allele 2 of +2018 of IL-1RN, allele 1 of +4845 of IL-1A, allele 3 of the 222/223 marker of IL-1A, allele 3 of the gz5/gz6 marker of IL-1A, allele 2 of the −889 marker of IL-1A, allele 2 of the +3954 marker of IL-1B, allele 1 of the −511 marker of IL-1B, allele 4 of the gaat.p33330 marker, allele 6 of the Y31 marker, allele 1 of +2018 of IL-1RN, allele 2 of +4845 of IL-1A, and allele 1 of the VNTR marker of IL-1RN.
 2. A kit comprising a means for detecting at least two alleles selected from the group consisting of: allele 2 of 1731 IL-RN, allele 2 of 1812 IL-1RN, allele 2 of 1868 IL-1RN, allele 2 of 1887 IL-1RN, allele 2 of 8006 IL-1RN, allele 2 of 8061 IL-1RN, allele 2 of 9589 IL-1RN, allele 4 of the 222/223 marker of IL-1A, allele 4 of the gz5/gz6 marker of IL-1A, allele 1 of the −889 marker of IL-1A, allele 1 of the +3954 marker of IL-1B, allele 2 of the −511 marker of IL-1B, allele 3 of the gaat.p33330 marker, allele 3 of the Y31 marker, allele 2 of +2018 of IL-1RN, allele 1 of +4845 of IL-1A, allele 3 of the 222/223 marker of IL-1A, allele 3 of the gz5/gz6 marker of IL-1A, allele 2 of the −889 marker of IL-1A, allele 2 of the +3954 marker of IL-1B, allele 1 of the -511 marker of IL-1B, allele 4 of the gaat.p33330 marker, allele 6 of the Y31 marker, allele 1 of +2018 of IL-1RN, allele 2 of +4845 of IL-1A, allele 2 of the VNTR marker of IL-1RN, and allele 1 of the VNTR marker of IL-1RN.
 3. The kit of claim 1 or 2, wherein said detecting means is selected from the group consisting of: a) allele specific oligonucleotide hybridization; b) size analysis; c) sequencing; d) hybridization; e) 5′ nuclease digestion; f) single-stranded conformation polymorphism; g) allele specific hybridization; h) primer specific extension; and j) oligonucleotide ligation assay.
 4. The kit of claim 1, further comprising a means for detecting allele 2 of the VNTR marker of IL-1RN.
 5. A kit comprising an isolated nucleic acid molecule which hybridizes to an allele selected from the group consisting of: allele 2 of 1731 IL-RN, allele 2 of 1812 IL-1RN, allele 2 of 1868 IL-1RN, allele 2 of 1887 IL-1RN, allele 2 of 8006 IL-1RN, allele 2 of 8061 IL-1RN, allele 2 of 9589 IL-1RN, allele 4 of the 222/223 marker of IL-1A, allele 4 of the gz5/gz6 marker of IL-1A, allele 1 of the −889 marker of IL-1A, allele 1 of the +3954 marker of IL-1B, allele 2 of the −511 marker of IL-1B, allele 3 of the gaat.p33330 marker, allele 3 of the Y31 marker, allele 2 of +2018 of IL-1RN, allele 1 of +4845 of IL-1A, allele 3 of the 222/223 marker of IL-1A, allele 3 of the gz5/gz6 marker of IL-1A, allele 2 of the −889 marker of IL-1A, allele 2 of the +3954 marker of IL-1B, allele 1 of the −511 marker of IL-1B, allele 4 of the gaat.p33330 marker, allele 6 of the Y31 marker, allele 1 of +2018 of IL-1RN, allele 2 of +4845 of IL-1A, allele 2 of the VNTR marker of IL-1RN, and allele 1 of the VNTR marker of IL-1RN.
 6. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 2 of 1731 IL-RN.
 7. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 2 of 1812 IL-1RN.
 8. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 2 of 1868 IL-1RN.
 9. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 2 of 1887 IL-1RN.
 10. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 2 of 8006 IL-1RN.
 11. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 2 of 8061 IL-1RN.
 12. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 2 of 9589 IL-1RN.
 13. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 4 of the 222/223 marker of IL-1A or allele 3 of the 222/223 marker of IL-1A.
 14. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 4 of the gz5/gz6 marker of IL-1A or allele 3 of the gz5/gz6 marker of IL-1A.
 15. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 1 of the −889 marker of IL-1A or allele 2 of the −889 marker of IL-1A.
 16. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 1 of the +3954 marker of IL-1B or allele 2 of the +3954 marker of IL-1B.
 17. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 2 of the −511 marker of IL-1B or allele 1 of the −511 marker of IL-1B.
 18. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 3 of the gaat.p33330 marker or allele 4 of the gaat.p33330 marker.
 19. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 3 of the Y31 marker or allele 6 of the Y31 marker.
 20. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 2 of +2018 of IL-1RN or allele 1 of +2018 of IL-1RN.
 21. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 1 of +4845 of IL-1A or allele 2 of +4845 of IL-1A.
 22. The kit of claim 5, wherein said isolated nucleic acid molecule hybridizes to the nucleotide corresponding to allele 2 of the VNTR marker of IL-1RN or allele 1 of the VNTR marker of IL-1RN. 